Kallikrein-inhibiting &#34;kunitz domain&#34; proteins and analogues thereof

ABSTRACT

Proteins are disclosed that are homologous to bovine pancreatic trypsin inhibitor (BPTI) Kunitz domains, and especially proteins that are homologous to lipoprotein-associated coagulation inhibitor (LACI) Kunitz domains, which inhibit one or more plasma and/or tissue kallikreins, and uses of such proteins in therapeutic and diagnostic methods also are disclosed. In particular, Kunitz domains derived from Kunitz domains of human origin and especially to the first Kunitz domain of LACI are disclosed.

This application is a 371 of PCT/US95/00299 which is continuation-in-part of U.S. application Ser. No. 08/208,264, filed Mar. 10, 1994 now abandoned, and is a continuation-in-part of application Ser. No. 08/179,904, filed Jan. 11, 1994, now abandoned, both of which application are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel classes of proteins and protein analogues which bind to and inhibit human plasma kallikrein.

2. Description of the Background Art

Kallikreins are serine proteases found in both tissues and plasma. Plasma kallikrein is involved in contact-activated (intrinsic pathway) coagulation, fibrinolysis, hypotension, and inflammation. (See BHOO92). These effects of kallikrein are mediated through the activities of three distinct physiological substrates: i) Factor XII (coagulation), ii) Pro-urokinase/plasminogen (fibrinolysis), and iii) Kininogens (hypotension and inflammation).

Kallikrein cleavage of kininogens results in the production of kinins, small highly potent bioactive peptides. The kinins act through cell surface receptors present on a variety of cell types. Intracellular heterotrimeric G-proteins link the kinin receptors to second messenger pathways including nitric oxide, adenyl cyclase, phospholipase A₂, and phospholipase C. Among the significant physiological activities of kinins are: (i) increased vascular permeability; (ii) vasodilation; (iii) bronchospasm; and (iv) pain induction. Thus, kinins mediate the life-threatening vascular shock and edema associated with bacteremia (sepsis) or trauma, the edema and airway hyperreactivity of asthma, and both inflammatory and neurogenic pain associated with tissue injury. The consequences of inappropriate plasma kallikrein activity and resultant kinin production are dramatically illustrated in patients with hereditary angioedema (HA). HA is due to a genetic deficiency of C1-inhibitor, the principal endogenous inhibitor of plasma kallikrein. Symptoms of HA include edema of the skin, subcutaneous tissues and gastrointestinal tract, and abdominal pain and vomiting. Nearly one-third of HA patients die by suffocation due to edema of the larynx and upper respiratory tract. Kallikrein is secreted as a zymogen (prekallikrein) that circulates as an inactive molecule until activated by a proteolytic event that frees the +NH₃ -IVGGTNSS . . . sequence of kallikrein (SEQ ID NO. 1). Human Plasma Prekallikrein is found in Genebank entry

Mature plasma Kallikrein contains 619 amino acids. Hydrolysis of the Arg₃₇₁ -Ile₃₇₂ peptide bond yields a two-chain proteinase joined by a disulfide bond. The amino-terminal light chain (248 residues) carries the catalytic site.

The main inhibitor of plasma kallikrein (pKA) in vivo is the C1 inhibitor; see SCHM87, pp.27-28. C1 is a serpin and forms an essentially irreversible complex with pKA. Although bovine pancreatic trypsin inhibitor (BPTI) was first said to be a strong pKA inhibitor with K_(i) =320 pM (AUER88), BERN93 indicates that its K_(i) for pKA is 30 nM (i.e., 30,000 pM). The G36S mutant had a K_(i) of over 500 nM. Thus, there is a need for a safe kallikrein inhibitor. The essential attributes of such an agent are:

i. Neutralization of relevant kallikrein enzyme(s);

ii. High affinity binding to target kallikreins to minimize dose;

iii. High specificity for kallikrein, to reduce side effects; and

iv. High degree of similarity to a human protein to minimize potential immunogenicity and organ/tissue toxicity.

The candidate target kallikreins to be inhibited are chymotrypsin-homologous serine proteases.

Excessive Bleeding

Excessive bleeding can result from deficient coagulation activity, elevated fibrinolytic activity, or a combination of the two. In most diatheses one must controll the activity of plasmin. However, plasma kallikrein (pKA) is an activator of plasminogen and a potent, selective pKA inhibitor may avert plasminogen activation. The clinically beneficial effect of BPTI in reducing blood loss is thought to result from its inhibition of plasmin (K_(D) ˜0.3 nM) or of plasma kallikrein (K_(D) ˜100 nM) or both enzymes. It has been found, however, that BPTI is sufficiently antigenic that second uses require skin testing. Furthermore, the doses of BPTI required to control bleeding are quite high and the mechanism of action is not clear. Some say that BPTI acts on plasmin while others say that it acts by inhibiting plasma kallikrein. FRAE89 reports that doses of about 840 mg of BPTI to 80 open-heart surgery patients reduced blood loss by almost half and the mean amount transfused was decreased by 74%. Miles Inc. has recently introduced Trasylol in the USA for reduction of bleeding in surgery (See Miles product brochure on Trasylol, which is hereby incorporated by reference.) LOHM93 suggests that plasmin inhibitors may be useful in controlling bleeding in surgery of the eye. SHER89 reports that BPTI may be useful in limiting bleeding in colonic surgery.

A kallikrein inhibitor that is much more potent than BPTI and that is almost identical to a human protein domain offers similar therapeutic potential, allows dose to be reduced, and poses less potential for antigenicity.

With recombinant DNA techniques, one may obtain a novel protein by expression of a mutated gene of a parental protein. Several strategies are known for picking mutations to test. One, "protein surgery", involves the introduction of one or more predetermined mutations within the gene of choice. A single polypeptide of completely predetermined sequence is expressed, and its binding characteristics are evaluated.

At the other extreme is random mutagenesis by means of relatively nonspecific mutagens such as radiation and various chemical agents, see Lehtovaara, E.P. Appln. 285,123, or by expression of highly degenerate DNA. It is also possible to follow an intermediate strategy in which some residues are kept constant, others are randomly mutated, and still others are mutated in a predetermined manner. This is called "variegation". See Ladner, et al. U.S. Pat. No. 5,220,409.

DENN94a and DENN94b report selections of Kunitz domains based on APP-I for binding to the complex of Tissue Factor with Factor VII_(a). They did not use LACI-K1 as parental and did not use pKA as a target. The highest affinity binder they obtained had K_(D) for their target of about 2 nM. Our first-round selectants for binding to pKA have affinity of about 0.3 nM, and our second round selectants are about at 0.1 nM (=100 pM) or better.

Proteins taken from a. particular species are assumed to be less likely to cause an immune response when injected into individuals of that species. Murine antibodies are highly antigenic in humans. "Chimeric" antibodies having human constant domains and murine variable domains are decidedly less antigenic. So called "humanized" antibodies have human constant domains and variable domains in which the CDRs are taken from murine antibodies while the framework of the variable domains are of human origin. "Humanized" antibodies are much less antigenic than are "chimeric" antibodies. In a "humanized" antibody, fifty to sixty residues of the protein are of non-human origin. The proteins of this invention comprise, in most cases, only about sixty amino acids and usually there are ten or fewer differences between the engineered protein and the parental protein. Although humans do develop antibodies even to human proteins, such as human insulin, such antibodies tend to bind weakly and the often do not prevent the injected protein from displaying its intended biological function. Using a protein from the species to be treated does not guarantee that there will be no immune response. Nevertheless, picking a protein very close in sequence to a human protein greatly reduces the risk of strong immune response in humans.

Kunitz domains are highly stable and can be produced efficiently in yeast or other host organisms. At least ten human Kunitz domains have been reported. Although BPTI was thought at one time to be a potent pKA inhibitor, there are, actually, no human Kunitz domains that inhibits pKA very well. Thus, it is a goal of this invention to provide sequences of Kunitz domain that are both potent inhibitors of pKA and close in sequence to human Kunitz domains.

The use of site-specific mutagenesis, whether nonrandom or random, to obtain mutant binding proteins of improved activity, is known in the art, but does not guarantee that the mutant proteins will have the desired target specificity or affinity. Given the poor anti-kallikrein activity of BPTI, mutation of BPTI or other Kunitz domain proteins would not have been considered, prior to this invention, a preferred method of obtaining a strong binder, let alone inhibitor, of kallikrein.

SUMMARY OF THE INVENTION

This invention relates to novel BPTI-homologous Kunitz domains, especially LACI homologues, which inhibit one or more plasma (and/or tissue) kallikreins, and to the therapeutic and diagnostic use of these novel proteins. In particular, this invention relates to Kunitz domains derived from Kunitz domains of human origin and especially to the first Kunitz domain of LACI; Kunitz domains of human origin are likely to be non-immunogenic in humans. The proteins of this invention inhibit plasma kallikrein (and/or tissue kallikrein) with a K_(D) of no more than 20 nM, preferably, no more than 5 nM, more preferably, no more than about 300 pM, and most preferably, no more than about 100 pM.

A specific, high affinity inhibitor of plasma kallikrein (and, where needed, tissue kallikrein) will demonstrate significant therapeutic utility in all pathological conditions mediated by kallikrein, and especially those associated with kinins. The therapeutic approach of inhibiting the catalytic production of kinins is considered preferable to antagonism of kinin receptors, since in the absence of kallikrein inhibition, receptor antagonists must compete with continuous kinin generation. Significantly, genetic deficiency of plasma kallikrein is benign and thus, inhibition of plasma kallikrein is likely to be safe. We have recently discovered a lead pKA inhibitor, designated KKII/3#6. This inhibitor is a variant of a naturally occurring human plasma protein Kunitz domain and demonstrates significantly greater kallikrein binding potency than Trasylol. KKII/3#6 has a K_(i) for kallikrein which is over 100 times that of both wild-type LACI and of BPTI, and is about 300 pM, In contrast, its K_(i) for plasmin is 10 μM, Proteins KK2/#11 and KK2/#13 are especially preferred pKA inhibitors and have K_(i) <300 pM and probably less than 100 pM. A reversible inhibitor is believed to be of greater utility than an irreversible inhibitor such as the C1 inhibitor.

Transfer of the subsequences that confer pKA binding into other Kunitz domains, particularly human Kunitz domains is disclosed.

The preferred pKA inhibitors of the present invention fullfil one or more of the following desiderata:

1) the inhibitor inhibits plasma kallikrein with a K_(i) no more than 20 nM, preferably 5 nM or less, more preferably 300 pM or less, and most preferably 100 pM or less,

2) the inhibitor comprises a Kunitz domain meeting the requirements shown in Table 14 with residues numbered by reference to BPTI,

3) the inhibitor has at the Kunitz domain positions 12-21 and 32-39 one of the amino-acid types listed for that position in Table 15, and

4) the inhibitor is substantially homologous to a reference sequence of essentially human origin selected from the group KKII/3#6, KK2/#11, KK2/#13, KK2/#1, KK2/#2, KK2/#3, KK2/#4, KK2/#6, KK2/#7, KK2/#8, KK2/#9, KK2/#10, KK2/#12, KK2conl, Human LACI-K2, Human LACI-K3, Human collagen μ3 KuDom, Human TFPI-2 DOMAIN 1, Human TFPI-2 DOMAIN 2, Human TFPI-2 DOMAIN 3, HUMAN ITI-K1, Human ITI-K2, HUMAN PROTEASE NEXIN-II, Human APP-I, DKI-1.2.1, DKI-1.3.1, DKI-2.1, DKI-3.1.1, DKI-3.2.1, DKI-3.3.1, DKI-4.1.1, DKI-4.2.1, DKI-4.2.2, DKI-5.1, and DKI-6.1

Nomenclature

Herein, affinities are stated as K_(D) (K_(D) (A,B)= A! B!/ A-B!). A numerically smaller K_(D) reflects higher affinity, For the purposes of this invention, a "kallikrein inhibiting protein" is one that binds and inhibits a specified kallikrein with K_(i) of about 20 nM or less. "Inhibition" refers to blocking the catalytic activity of kallikrein and so is measurable in vitro in assays using chromogenic or fluorogenic substrates or in assays involving macromolecules.

Amino-acid residues are discussed in three ways: full name of the amino acid, standard three-letter code, and standard single-letter code. The text uses full names and three-letter code where clarity requires.

    ______________________________________                                         A = Ala     G = Gly   M = Met    S = Ser                                       C = Cys     H = His   N = Asn    T = Thr                                       D = Asp     I = Ile   P = Pro    V = Val                                       E = Glu     K = Lys   Q = Gln    W = Trp                                       F = Phe     L = Leu   R = Arg    Y = Tyr                                       ______________________________________                                    

For the purposed of this invention, "substantially homologous" sequences are at least 51%, more preferably at least 80%, identical, over any specified regions. For this invention, "substantially homologous" includes exact identity. Sequences would still be "substantially homologous" if within one region of at least 20 amino acids they are sufficiently similar (51% or more) but outside the region of comparison they differed totally. An insertion of one amino acid in one sequence relative to the other counts as one mismatch. Most preferably, no more than six residues, other than at termini, are different. Preferably, the divergence in sequence, particularly in the specified regions, is in the form of "conservative modifications".

"Conservative modifications" are defined as

(a) conservative substitutions of amino acids as defined in Table 9; and

(b) single or multiple insertions or deletions of amino acids at termini, at domain boundaries, in loops, or in other segments of relatively high mobility.

Preferably, except at termini, no more than about six amino acids are inserted or deleted at any locus, and the modifications are outside regions known to contain important binding sites.

Kunitz Domains

Herein, "Kunitz domain" and "KuDom" are used interchangeably to mean a homologue of BPTI (not of the Kunitz soya-bean trypsin inhibitor). A KuDom is a domain of a protein having at least 51 amino acids (and up to about 61 amino acids) containing at least two, and preferably three, disulfides. Herein, the residues of all Kunitz domains are numbered by reference to BPTI (i.e. residues 1-58, amino-acid sequence in Table 2). Thus the first cysteine residue is residue 5 and the last cysteine is 55. An amino-acid sequence shall, for the purposes of this invention, be deemed a Kunitz domain if it can be aligned, with three or fewer mismatches, to the sequence shown in Table 14. An insertion or deletion of one residue shall count as one mismatch. In Table 14, "x" matches any amino acid and "X" matches the types listed for that position Disulfide bonds link at least two of: 5 to 55, 14 to 38, and 30 to 51. The number of disulfides may be reduced by one, but none of the standard cysteines shall be left unpaired. Thus, if one cysteine is changed, then a compensating cysteine is added in a suitable location or the matching cysteine is also replaced by a non-cysteine (the latter being generally preferred). For example, Drosophila funebris male accessory gland protease inhibitor has no cysteine at position 5, but has a cysteine at position -1 (just before position 1); presumably this forms a disulfide to CYS₅₅. If Cys₁₄ and Cys₃₈ are replaced, the requirement of Gly,₁₂, (Gly or Ser)₃₇, and Gly₃₆ are dropped. From zero to many residues, including additional domains (including other KuDoms), can be attached to either end of a Kunitz domain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Protease inhibitors, such as Kunitz domains, function by binding into the active site of the protease so that a peptide bond (the "scissile bond") is: 1) not cleaved, 2) cleaved very slowly, or 3) cleaved to no effect because the structure of the inhibitor prevents release or separation of the cleaved segments. In Kunitz domains, disulfide bonds act to hold the protein together even if exposed peptide bonds are cleaved. From the residue on the amino side of the scissile bond, and moving away from the bond, residues are conventionally called P1, P2, P3, etc. Residues that follow the scissile bond are called P1', P2', P3', etc. (SCHE67, SCHE68). It is generally accepted that each serine protease has sites (comprising several residues) S 1, S2, etc. that receive the side groups and main-chain atoms of residues P1, P2, etc. of the substrate or inhibitor and sites S1', S2', etc. that receive the side groups and main-chain atoms of P1', P2', etc. of the substrate or inhibitor. It is the interactions between the S sites and the P side groups and main chain atoms that give the protease specificity with respect to substrates and the inhibitors specificity with respect to proteases. Because the fragment having the new amino terminus leaves the protease first, many worker designing small molecule protease inhibitors have concentrated on compounds that bind sites S1, S2, S3, etc.

LASK80 reviews protein protease inhibitors. Some inhibitors have several reactive sites on one polypeptide chain, and these domains usually have different sequences, specificities, and even topologies. It is known that substituting amino acids in the P₅ to P₅ ' region influences the specificity of an inhibitor. Previously, attention has been focused on the P1 residue and those very close to it because these can change the specificity from one enzyme class to another. LASK80 suggests that among KuDoms, inhibitors with P1=Lys or Arg inhibit trypsin, those with P1=Tyr, Phe, Trp, Leu and Met inhibit chymotrypsin, and those with P1=Ala or Ser are likely to inhibit elastase. Among the Kazal inhibitors, LASK80 continues, inhibitors with P1=Leu or Met are strong inhibitors of elastase, and in the Bowman-Kirk family elastase is inhibited with P1=Ala, but not with P1=Leu. Such limited changes do not provide inhibitors of truly high affinity (i.e. better than 1 to 10 nM).

KuDoms are defined above. The 3D structure (at high resolution) of BPTI (the archetypal Kunitz domain) is known. One of the X-ray structures is deposited in the Brookhaven Protein Data Bank as "6PTI"!. The 3D structure of some BPTI homologues (EIGE90, HYNE90) are known. At least seventy KuDom sequences are known. Known human homologues include three KuDoms of LACI (WUNT88, GERA89, NOVO89), two KuDoms of Inter-α-Trypsin Inhibitor, APP-I (KIDO88), a KuDom from collagen, and three KuDoms of TFPI-2 (SPRE94).

LACI

Lipoprotein-associated coagulation inhibitor (LACI) is a human serum phosphoglycoprotein with a molecular weight of 39 kDa (amino-acid sequence in Table 1) containing three KuDoms. We refer hereinafter to the protein as LACI and to the Kunitz domains thereof as LACI-K1 (residues 50 to 107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to 270). The cDNA sequence of LACI is reported in WUNT88. GIRA89 reports mutational studies in which the P1 residues of each of the three KuDoms were altered. LACI-K1 inhibits Factor VIIa (F.VII_(a)) when F.VII_(a) is complexed to tissue factor and LACI-K2 inhibits Factor X_(a). It is not known whether LACI-K3 inhibits anything. Neither LACI nor any of the KuDoms of LACI is a potent plasma kallikrein inhibitor.

In one preferred embodiment of this invention, KuDoms are substantially homologous with LACI-K1, but differ in ways that confer strong plasma kallikrein inhibitory activity discussed below. Other KuDoms of this invention are homologous to other naturally-occurring KuDoms, particularly to other human KuDoms. For use in humans, the proteins of this invention are designed to be highly similar in sequence to one or another human KuDom to reduce the risk of causing an immune response.

Variegation of a protein is typically achieved by preparing a correspondingly variegated mixture of DNA (with variable codons encoding variable residues), cloning it into suitable vectors, and expressing the DNA in suitable host cells. For any given protein molecule of the library, the choice of amino acid at each variable residue, subject to the above constraints, is random, the result of the happenstance of which DNA expressed that protein molecule.

FIRST LACI-K1 LIBRARY SCREENED FOR pKA BINDING

Applicants have screened a first large library of LACI-K1 domains (patern of variegation is shown in Table 21), with the results shown in Table 3. In Table 3, "Library Residues" are those permitted to occur, randomly, at that position, in the library, and "Preferred Residues" are those appearing at that position in at least one of the 10 variants identified as binding to human kallikrein.

At residues 13, 16, 17, 18, 31, and 32, the selections are very strong. At position 34, the selection for either SER or THR is quite strong. At position 39, the selection for GLY is strong. Position 19 seems to be rather tolerant.

It should be appreciated that Applicants have not sequenced all of the positive isolates in this or other the libraries herein disclosed, that some of the possible mutant proteins may not have been present in the library in detectable amounts, and that, at some positions, only some of the possible amino acids were intended to be included in the library.

SECOND LIBRARY OF LACI-K1 and SELECTION OF NEW KALLIKREIN INHIBITORS

Applicants prepared a second LACI-K1 library as shown in Table 750. This library utilized the observation of the first selection and allows variability at positions 10, 11, 13, 15, 16, 17, 18, 19, and 21. The residues at positions 34 and 39 were fixed at S₃₄ and G₃₉. Selectants KK2/#1 through KK2/#13, as shown in Table 2 were obtained in the same manner as described in the Example section for the first screeneing. Applicants prepared the proteins KK2/#11 and KK2/#13 in S. cerevisiae in the Matα system described herein. Preliminary measurements indicate that these proteins are very potent pKA inhibitors with K less than 300 pM and probably less than 100 pM.

Using the selected sequences and the binding data of selected KuDoms, we can write a recipe for a high-affinity pKA-inhibiting KuDom that can be applied to other human KuDom parentals. First, the KuDom must meet the requirements in Table 14. The substitutions shown in Table 15 are likely to confer high-affinity pKA inhibitory activity on any KuDom. Thus a protein that contains a sequence that is a KuDom, as shown in Table 14, and that contains at each of the position 12-21 and 32-39 an amino-acid type shown in Table 15 for that position is likely to be a potent inhibitor of human pKA. More preferably, the protein would have an amino-acid type shown in Table 15 for all of the positions listed in Table 15. To reduce the potential for immune response, one should use one or another human KuDom as parental protein to give the sequence outside the binding region.

It is likely that a protein that comprises an amino-acid sequence that is substantially homologous to one of KK2/#13, KK2/#11, or KKII/3#6 from residue 5 through residue 55 (as shown in Table 2) and is identical to one of KK2/#13, KK2/#11, or KKII/3#6 at positions 13-19, 31, 32, 34, and 39 will inhibit human pKA with a K_(i) of 5 nM or less. KK2/#13, KK2/#11, and KKII/3#6 differs from LACI-K1 at 10, 8, and 7 positions respectively. It is not clear that these substitutions are equally important in fostering pKA binding and inhibition. From the known pKA inhibitors listed, one can prepare a series of molecules that are progressively reverted toward LACI-K1. It is expected that the molecules will show less affinity for pKA but also less potential for antigenicity. A person skilled in the art can pick a protein of sufficient potency and low immunogenicity from this collection. It is also possible that substitutions in one of the listed pKA inhibitors by amino acids that differ from LACI-K1 can reduce the immunogenicity without reducing the affinity for pKA to a degree that makes the protein unsuitable for use as a drug.

DESIGNED KuDom PKA Inhibitors

Hereinafter, "DKI" will mean a "Designed PKA Inhibitor" that are KuDoms that incorporate amino-acid sequence information from the SPI series of molecules, especially KK2/#13, KK2/#11, or KKII/3#6. Sequences of several DKIs and their parental proteins are given in Table 2. Hereinafter, the statement "the mutations X_(nn) Y₁, X_(nn) Y₂, . . . may not be needed" means that each of the mutations might be separately found to be unnecessary. That is, the list is not to be taken as a block to be applied together, but as a list of things to be tested. Similarly, the lists of additional mutations are to be tested singly.

Protein DKI-1.2.1 is based on human LACI-K2 and shown in Table 2. The mutations P11G, I13R, Y17A, I18H, T19P, Y21W, R32E, K34S, and L39G are likely to confer high affinity for pKA. Some of these substitutions may not be necessary; in particular, P11G and T19P may not be necessary. Other mutations that might improve the pKA affinity include E9A, D10E, G16A, Y21F, and L39E.

Protein DKI-1.3.1 (Table 2) is based on human LACI-K3. The mutations R11D, L13P, N17A, E18H, N19P, R31E, K34S, and S36G are intended to confer high affinity for pKA. Some of these substitutions may not be necessary; in particular, N19P may not be necessary. Other changes that might improve K_(D) include D10E, F21W and G39E.

Protein DKI-2.1 (Table 2) is a based on the human collagen α3 KuDom. The mutations D16A, F17A, I18H, R32E, and W34S are likely to confer high affinity for pKA. Some of these substitutions may not be necessary; in particular, R32E may not be necessary. Other mutations that might improve the pKA affinity include K9A, D10E, D 16G, K20R, R32T, W34V, and G39E.

DKI-3.1.1 (Table 2) is derived from Human TFPI-2 domain 1. The exchanges Y11G, L17A, L18H, R31E, and L34S are likely to confer high affinity for pKA. The mutation L34S may not be needed. Other mutations that might foster pKA binding include Y21W, Y21F, Q32E, L34T, L34I, and E39G.

DKI-3.2.1 (Table 2) is derived from Human TFPI-2 domain 2. This parental domain contains insertions after residue 9 (one residue) and 42 (two residues). The mutations E15R, G16A, S 17A, T18H,E19P, K32T, and F34V are intended to confer affinity for pKA. If one needs a pKA inhibitor based on TFPI domain 2, a preferred route is to make a library of domains allowing the substitutions given and many others and then select binders.

DKI-3.3.1 (Table 2) is derived from human TFPI-2, domain 3. The substitutions L13H, S 15R, and N17A are likely to confer high affinity for pKA. Other mutations that might foster pKA binding include D10E, T19Q, Y21W, T36G, and G39E.

DKI-4.1.1 (Table 2) is from human ITI-K1 by assertion of S10D, M15R, M17A, T18H, Q34S, and M39G. The mutations M39G and Q34V may not be necessary. Other mutations that should foster pKA binding include: G16A, M17N, S19Q, Y21W, and Y2IF.

DKI-4.2.1 (Table 2) is from human ITI-K2 through the mutations V10D, R11D, F17A, I18H, V31E, L32E, P34S, and Q39E. The mutations V31E, L32E, and Q39E might not be necessary. Other mutation that should foster pKA binding include: V10E, Q19P, L20R, W21F, P34I, and Q39G. DKI-4.2.2 has eight mutations: V10D, R11D, F17A, I18H, L20R, V31E, L32E, and P34S.

DKI-5.1 is derived from human APP-I (also known as Protease Nexin-II) by mutations M17A, I18H, S19P, A31E, and P32E and is likely to be a potent pKA inhibitor. The mutations S19P, A3 1E, and P32E many not be needed. Other mutations that might foster pKA binding include T11D.

DKI-6.1 is derived from the HKI B9 KuDom (NORR93) by the five substitutions: K11D, Q15R, T16A, M17A, M18H, T19P, and L32E. DKI-6.1 is likely to be a potent pKA inhibitor. The mutations L32E, and T19P might not be needed.

Although BPTI is not an especially good pKA inhibitor, it could be made into one. DKI-7.1 is derived from BPTI by the mutations Y10E, K15R, R17A, RI18H, I19P, Q31E, T32E, and R39E which is likely to increase the affinity for pKA. The mutations Y10E, K15R, I19P, Q31E, T32E, and R39E may not be needed; the really important mutations are R17A and RI18H.

MODIFICARION OF KUNITZ DOMAINS

KuDoms are quite small; if this should cause a pharmacological problem, such as excessively quick elimination from circulation, two or more such domains may be joined. A preferred linker is a sequence of one or more amino acids. A preferred linker is one found between repeated domains of a human protein, especially the linkers found in human BPTI homologues, one of which has two domains (BALD85, ALBR83a, ALBR83b) and another of which has three (WUNT88). Peptide linkers have the advantage that the entire protein may then be expressed by recombinant DNA techniques. It is also possible to use a nonpeptidyl linker, such as one of those commonly used to form immunogenic conjugates. An alternative means of increasing the serum residence of a BPTI-like KuDom is to link it to polyethyleneglycol, so called PEGylation (DAVI79).

WAYS TO IMPROVE SPECIFICITY OF, FOR EXAMPLE, KKII/3#7, KK2/#11, AND KK2/#13 FOR PLASMA KALLIKREIN:

Because we have made a large part of the surface of KKII/3#6, KK2/#11, and KK2/#13 complementary to the surface of pKA, R,₁₅ is not essential for specific binding to pKA. Many of the enzymes in the clotting and fibrinolytic pathways cut preferentially after Arg or Lys. Not having a basic residue at the P1 position may give rise to greater specificity. The variant KKII/3#7-K15A (shown in Table 27), having an ALA at P1, is likely to be a good pKA inhibitor and may have higher specificity for pKA relative to other proteases than doesKKII/3#7. The affinity of KKII/3#7-K15A for pKA is likely to be less than the affinity of KKII/3#7 for pKA, but the loss of affinity for other Arg/Lys-preferring enzymes is likely to be greater and, in many applications, specificity is more important than affinity. Other mutants that are likely to have good affinity and very high specificity include KK2/#13-R15A and KK2/#11-R15S. This approach could be applied to other high-affinity pKA inhibitors.

MODE OF PRODUCTION

The proteins of this invention may be produced by any conventional technique, including

(a) nonbiological synthesis by sequential coupling of component amino acids,

(b) production by recombinant DNA techniques in a suitable host cell, and

(c) removal of undesired sequences from LACI and coupling of synthetic replacement sequences

The proteins disclosed herein are preferably produced, recombinantly, in a suitable host, such as bacteria from the genera Bacillus, Escherichia, Salmonella, Erwinia, and yeasts from the genera Hansenula, Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and Schizosaccharomyces, or cultured mammalian cells such as COS-1. The more preferred hosts are microorganisms of the species Pichia pastoris, Bacillus subtilis, Bacillus brevis, Saccharomyces cerevisiae, Escherichia coli and Yarrowia lipolytica. Any promoter, regulatable or constitutive, which is functional in the host may be used to control gene expression.

Preferably the proteins are secreted. Most preferably, the proteins are obtained from conditioned medium. It is not required that the proteins described herein be secreted. Secretion is the preferred route because proteins are more likely to fold correctly, can be produced in conditioned medium with few contaminants, and are less likely to be toxic to host cells. Secretion is not required.

Unless there is a specific reason to include glycogroups, we prefer proteins designed to lack N-linked glycosylation sites to reduce potential for antigenicity of glycogroups and so that equivalent proteins can be expressed in a wide variety of organisms including: 1) E. coli, 2) B. subtilis, 3) P. pastoris, 4) S. cerevisiae, and 5) mammalian cells.

Several means exist for reducing the problem of host cells producing proteases that degrade the recombinant product; see, inter alia BANE90 and BANE91. VAND92 reports that overexpression of the B. subtilis signal peptidase in E. coli, leads to increased expression of a heterologous fusion protein. ANBA88 reports that addition of PMSF (a serine proteases inhibitor) to the culture medium improved the yield of a fusion protein.

Other factors that may affect production of these and other proteins disclosed herein include: 1) codon usage (optimizing codons for the host is preferred), 2) signal sequence, 3) amino-acid sequence at intended processing sites, presence and localization of processing enzymes, deletion, mutation, or inhibition of various enzymes that might alter or degrade the engineered product and mutations that make the host more permissive in secretion (permissive secretion hosts are preferred).

Reference works on the general principles of recombinant DNA technology include Watson et al., Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif. (1987); Darnell et al., Molecular Cell Biology, Scientific American Books, Inc., New York, N.Y. (1986); Lewin, Genes II, John Wiley & Sons, New York, N.Y. (1985); Old, et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, Berkeley, Calif. (1981); Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989); and Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, N.Y., (1987, 1992). These references are herein entirely incorporated by reference as are the references cited therein.

PREPARATION OF PEPTIDES

Chemical polypeptide synthesis is a rapidly evolving area in the art, and methods of solid phase polypeptide synthesis are well-described in the following references, hereby entirely incorporated by reference: (Merrifield, J Amer Chem Soc 85:2149-2154 (1963); Merrifield, Science 232:341-347 (1986); Wade et al., Biopolymers 25:S21-S37 (1986); Fields, Int J Polypeptide Prot Res 35:161 (1990); MilliGen Report Nos. 2 and 2a, Millipore Corporation, Bedford, Mass, 1987) Ausubel et al, supra, and Sambrook et al, supra. Tan and Kaiser (Biochemistry, 1977, 16:1531-41) synthesized BPTI and a homologue eighteen years ago.

As is known in the art, such methods involve blocking or protecting reactive functional groups, such as free amino, carboxyl and thio groups. After polypeptide bond formation, the protective groups are removed. Thus, the addition of each amino acid residue requires several reaction steps for protecting and deprotecting. Current methods utilize solid phase synthesis, wherein the C-terminal amino acid is covalently linked to an insoluble resin particles that can be filtered. Reactants are removed by washing the resin particles with appropriate solvents using an automated machine. Various methods, including the "tBoc" method and the "Fmoc" method are well known in the art. See, inter alia, Atherton et al., J Chem Soc Perkin Trans 1:538-546 (1981) and Sheppard et al, Int J Polypeptide Prot Res 20:451-454 (1982).

ASSAYS FOR PLASMA KALLIKREIN BINDING AND INHIBITION

Any suitable method may be used to test the compounds of this invention. Scatchard (Ann NY Acad Sci (1949) 51:660-669) described a classical method of measuring and analyzing binding which is applicable to protein binding. This method requires relatively pure protein and the ability to distinguish bound protein from unbound.

A second appropriate method of measuring K_(D) is to measure the inhibitor activity against the enzyme. If the K_(D) to be measured is in the 1 nM to 1 μM range, this method requires chromogenic or fluorogenic substrates and tens of micrograms to milligrams of relatively pure inhibitor. For the proteins of this invention, having K_(D) in the range 5 nM to 50 pM, nanograms to micrograms of inhibitor suffice. When using this method, the competition between the inhibitor and the enzyme substrate can give a measured K_(i) that is higher than the true K_(i). Measurement reported here are not so corrected because the correction would be very small and the any correction would reduce the K_(i). Here, we use the measured K_(i) as a direct measure of K_(D).

A third method of determining the affinity of a protein for a second material is to have the protein displayed on a genetic package, such as M13, and measure the ability of the protein to adhere to the immobilized "second material". This method is highly sensitive because the genetic packages can be amplified. We obtain at least semiquantitative values for the binding constants by use of a pH step gradient. Inhibitors of known affinity for the protease are used to establish standard profiles against which other phage-displayed inhibitors are judged. Any other suitable method of measuring protein binding may be used.

Preferably, the proteins of this invention have a K_(D) for pKA of at most about 5nM, more preferably at most about 300 pM, and most preferably 100 pM or less. Preferably, the binding is inhibitory so that K_(i) is the same as K_(D). The K_(i) of KKII/3#6 is about 300 pM and the K_(i) s of KK2/#11 and KK2/#13 are less than 300 pM and probably less than 100 pM.

Pharmaceutical Methods and Preparations

The preferred subject of this invention is a mammal. The invention is particularly useful in the treatment of humans, but is suitable for veternary applications too.

Herein, "protection" includes "prevention", "suppression", and "treatment". "Prevention" involves administration of drug prior to the induction of disease. "Suppression" involves administration of drug prior to the clinical appearance of disease. "Treatment" involves administration of drug after the appearance of disease.

In human and veterinary medicine, it may not be possible to distinguish between "preventing" and "suppressing" since the inductive event(s) may be unknown or latent, or the patient is not ascertained until after the occurrence of the inductive event(s). We use the term "prophylaxis" as distinct from "treatment" to encompass "preventing" and "suppressing". Herein, "protection" includes "prophylaxis". Protection need not be absolute to be useful.

Proteins of this invention may be administered, by any means, systemically or topically, to protect a subject against a disease or adverse condition. For example, administration of such a composition may be by any parenteral route, by bolus injection or by gradual perfusion. Alternatively, or concurrently, administration may be by the oral route. A suitable regimen comprises administration of an effective amount of the protein, administered as a single dose or as several doses over a period of hours, days, months, or years.

The suitable dosage of a protein of this invention may depend on the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the desired effect. However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation by adjustment of the dose in ways known in the art.

For methods of pre clinical and clinical testing of drugs, including proteins, see, e.g., Berkow et al, eds., The Merck Manual, 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et al, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985), which references and references cited there are hereby incorporated by reference.

In addition to a protein here disclosed, a pharmaceutical composition may contain pharmaceutically acceptable carriers, excipients, or auxiliaries. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra.

In Vitro Diagnostic Methods and Reagents

Proteins of this invention may be applied in vitro to any suitable sample that might contain plasma kallikrein to measure the pKA present. To do so, the assay must include a Signal Producing System (SPS) providing a detectable signal that depends on the amount of pKA present. The signal may be detected visually or instrumentally. Possible signals include production of colored, fluorescent, or luminescent products, alteration of the characteristics of absorption or emission of radiation by an assay component or product, and precipitation or agglutination of a component or product.

The component of the SPS most intimately associated with the diagnostic reagent is called the "label". A label may be, e.g., a radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound, or an agglutinable particle. A radioactive isotope can be detected by use of, for example, a γ counter or a scintillation counter or by autoradiography. Isotopes which are particularly useful are ³ H, ¹²⁵ I, ¹³¹ I, ³⁵ S, ¹⁴ C, and, preferably, ¹²⁵ I. It is also possible to label a compound with a fluorescent compound. When the fluorescently labeled compound is exposed to light of the proper wave length, its presence can be detected. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine. Alternatively, fluorescence-emitting metals, such as ¹²⁵ Eu or other lanthanide, may be attached to the binding protein using such metal chelating groups as diethylenetriaminetetraacetic acid or ethylenediamine-tetraacetic acid. The proteins also can be detectably labeled by coupling to a cheminuminescent compound, such as luminol, isolumino, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Likewise, a bioluminescent compound, such as luciferin, luciferase and aequorin, may be used to label the binding protein. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Enzyme labels, such as horseradish peroxidase and alkaline phosphatase, are preferred.

There are two basic types of assays: heterogeneous and homogeneous. In heterogeneous assays, binding of the affinity molecule to analyte does not affect the label; thus, to determine the amount of analyte, bound label must be separated from free label. In homogeneous assays, the interaction does affect the activity of the label, and analyte can be measured without separation.

In general, a kallikrein-binding protein (KBP) may be used diagnostically in the same way that an anti-pKA antibody is used. Thus, depending on the assay format, it may be used to assay pKA, or, by competitive inhibition, other substances which bind pKA.

The sample will normally be a biological fluid, such as blood, urine, lymph, semen, milk, or cerebrospinal fluid, or a derivative thereof, or a biological tissue, e.g., a tissue section or homogenate. The sample could be anything. If the sample is a biological fluid or tissue, it may be taken from a human or other mammal, vertebrate or animal, or from a plant. The preferred sample is blood, or a fraction or derivative thereof

In one embodiment, the pKA-binding protein (KBP) is immobilized, and pKA in the sample is allowed to compete with a known quantity of a labeled or specifically labelable pKA analogue. The "pKA analogue" is a molecule capable of competing with pKA for binding to the KBP, which includes pKA itself It may be labeled already, or it may be labeled subsequently by specifically binding the label to a moiety differentiating the pKA analogue from pKA. The phases are separated, and the labeled pKA analogue in one phase is quantified.

In a "sandwich assay", both an insolubilized pKA-binding agent (KBA), and a labeled KBA are employed. The pKA analyte is captured by the insolubilized KBA and is tagged by the labeled KBA, forming a tertiary complex. The reagents may be added to the sample in any order. The KBAs may be the same or different, and only one KBA need be a KBP according to this invention (the other may be, e.g., an antibody). The amount of labeled KBA in the tertiary complex is directly proportional to the amount of pKA in the sample.

The two embodiments described above are both heterogeneous assays. A homogeneous assay requires only that the label be affected by the binding of the KBP to pKA. The pKA analyte may act as its own label if a pKA inhibitor is used as a diagnostic reagent.

A label may be conjugated, directly or indirectly (e.g., through a labeled anti-KBP antibody), covalently (e.g, with SPDP) or noncovalently, to the pKA-binding protein, to produce a diagnostic reagent. Similarly, the pKA binding protein may be conjugated to a solid phase support to form a solid phase ("capture") diagnostic reagent. Suitable supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, and magnetite. The carrier can be soluble to some extent or insoluble for the purposes of this invention. The support material may have any structure so long as the coupled molecule is capable of binding pKA.

In Vivo Diagnostic Uses

A Kunitz domain that binds very tightly to pKA can be used for in vivo imaging. Diagnostic imaging of disease foci was considered one of the largest commercial opportunities for monoclonal antibodies, but this opportunity has not been achieved. Despite considerable effort, only two monoclonal antibody-based imaging agents have been approved. The disappointing results obtained with monoclonal antibodies is due in large measure to:

i) Inadequate affinity and/or specificity;

ii) Poor penetration to target sites;

iii) Slow clearance from nontarget sites;

iv) Immunogenicity (most are murine); and

v) High production cost and poor stability.

These limitations have led most in the diagnostic imaging field to begin to develop peptide-based imaging agents. While potentially solving the problems of poor penetration and slow clearance, peptide-based imaging agents are unlikely to possess adequate affinity, specificity and in vivo stability to be useful in most applications.

Engineered proteins are uniquely suited to the requirements for an imaging agent. In particular the extraordinary affinity and specificity that is obtainable by engineering small, stable, human-origin protein domains having known in vivo clearance rates and mechanisms combine to provide earlier, more reliable results, less toxicity/side effects, lower production and storage cost, and greater convenience of label preparation. Indeed, it should be possible to achieve the goal of realtime imaging with engineered protein imaging agents. Thus, a Kallikrein-binding protein, e.g., KKII/3#6, KK2/#11, and KK2/#13 may be used for localizing sites of excessive pKA activity.

Radio-labelled binding protein may be administered to the human or animal subject. Administration is typically by injection, e.g., intravenous or arterial or other means of administration in a quantity sufficient to permit subsequent dynamic and/or static imaging using suitable radio-detecting devices. The dosage is the smallest amount capable of providing a diagnostically effective image, and may be determined by means conventional in the art, using known radio-imaging agents as guides.

Typically, the imaging is carried out on the whole body of the subject, or on that portion of the body or organ relevant to the condition or disease under study. The radio-labelled binding protein has accumulated. The amount of radio-labelled binding protein accumulated at a given point in time in relevant target organs can then be quantified.

A particularly suitable radio-detecting device is a scintillation camera, such as a γ camera. The detection device in the camera senses and records (and optional digitizes) the radioactive decay. Digitized information can be analyzed in any suitable way, many of which are known in the art. For example, a time-activity analysis can illustrate uptake through clearance of the radio-labelled binding protein by the target organs with time.

Various factors are taken into consideration in picking an appropriate radioisotope. The isotope is picked: to allow good quality resolution upon imaging, to be safe for diagnostic use in humans and animals, and, preferably, to have a short half-life so as to decrease the amount of radiation received by the body. The radioisotope used should preferably be pharmacologically inert, and the quantities administered should not have substantial physiological effect. The binding protein may be radio-labelled with different isotopes of iodine, for example ¹²³ I, ¹²⁵ I, or ¹³¹ I (see, for example, U.S. Pat. No. 4,609,725). The amount of labeling must be suitably monitored.

In applications to human subjects, it may be desirable to use radioisotopes other than ¹²⁵ I for labelling to decrease the total dosimetry exposure of the body and to optimiize the detectability of the labelled molecule. Considering ready clinical availability for use in humans, preferred radio-labels include: ^(99m) Tc, ⁶⁷ Ga, ⁶⁸ Ga, ⁹⁰ Y, ¹¹¹ In, ^(113m) In, ¹²³ I,¹⁸⁶ Re, ¹⁸⁸ Re or ²¹¹ At. Radio-labelled protein may be prepared by various methods. These include radio-halogenation by the chloramine-T or lactoperoxidase method and subsequent purification by high pressure liquid chromatography, for example, see Gutkowska et al in "Endocrinology and Metabolism Clinics of America: (1987) 16 (1): 183. Other methods of radio-labelling can be used, such as IODOBEADS™.

A radio-labelled protein may be administered by any means that enables the active agent to reach the agent's site of action in a mammal. Because proteins are subject to digestion when administered orally, parenteral administration, i.e., intravenous subcutaneous, intramuscular, would ordinarily be used to optimize absorption.

High-affinity, high-specificity inhibitors are also useful for in vitro diagnostics of excess human pKA activity.

Other Uses

The kallikrein-binding proteins of this invention may also be used to purify kallikrein from a fluid, e.g., blood. For this, the KBP is preferably immobilized on a support. Such supports, include those already mentioned as useful in preparing solid phase diagnostic reagents.

Proteins can be used as molecular weight markers for reference in the separation or purification of proteins. Proteins may need to be denatured to serve as molecular weight markers. A second general utility for proteins is the use of hydrolyzed protein as a nutrient source. Proteins may also be used to increase the viscosity of a solution.

The proteins of this invention may be used for any of the foregoing purposes, as well as for therapeutic and diagnostic purposes as discussed further earlier in this specification.

EXAMPLE 1: Construction of First LACI-K1 Library

A synthetic oligonucleotide duplex having NsiI- and MluI-compatible ends was cloned into a parental vector (LACI:III) previously cleaved with the above two enzymes. The resultant ligated material was transfected by electroporation into XLIMR (F⁻) Escherichia coli strain and plated on Amp plates to obtain phage-generating Ap^(R) colonies. The variegation scheme for Phase 1 focuses on the P1 region, and affected residues 13, 16, 17, 18 and 19. It allowed for 6.6×10⁵ different DNA sequences (3.1×10⁵ different protein sequences). The library obtained consisted of 1.4×10⁶ independent cfu's which is approximately a two fold representation of the whole library. The phage stock generated from this plating gave a total titer of 1.4×10¹³ pfu's in about 3.9 ml, with each independent clone being represented, on average, 1×10⁷ in total and 2.6×10⁶ times per ml of phage stock.

To allow for variegation of residues 31, 32, 34 and 39 (phase II), synthetic oligonucleotide 5 duplexes with MluI- and BstEII- compatible ends were cloned into previously cleaved R_(f) DNA derived from one of the following

i) the parental construction,

ii) the phase I library, or

iii) display phage selected from the first phase binding to a given target.

The variegation scheme for phase II allows for 4096 different DNA sequences (1600 different protein sequences) due to alterations at residues 31, 32, 34 and 39. The final phase II variegation is dependent upon the level of variegation remaining following the three rounds of binding and elution with a given target in phase I.

The combined possible variegation for both phases equals 2.7×10⁸ different DNA sequences or 5.0×10⁷ different protein sequences. When previously selected display phage are used as the origin of R_(f) DNA for the phase II variegation, the final level of variegation is probably in the range of 10⁵ to 10⁶.

EXAMPLE 2: Screening of LACI (K1) Library for Binding to Kallikrein

The overall scheme for selecting a LACI-K1 variant to bind to a given protease involves incubation of the phage-display library with the kallikrein-beads of interest in a buffered solution (PBS containing 1 mg/ml BSA) followed by washing away the unbound and poorly retained display-phage variant with PBS containing 0.1% Tween 20. Kallikrein beads were made by coupling human plasma Kallikrein (Calbiochem, San Diego, Calif., # 420302) to agarose beads using Reactigel (6x) (Pierce, Rockford, II, #202606). The more strongly bound display-phage are eluted with a low pH elution buffer, typically citrate buffer (pH 2.0) containing 1 mg/ml BSA, which is immediately neutralized with Tris buffer to pH 7.5. This process constitutes a single round of selection.

The neutralized eluted display-phage can be either used:

i) to inoculate an F⁺ strain of E. coli to generate a new display-phage stock, to be used for subsequent rounds of selection (so-called conventional screening), or

ii) be used directly for another immediate round of selection with the protease beads (so-called quick screening).

Typically, three rounds of either method, or a combination of the two, are performed to give rise to the final selected display-phage from which a representative number are sequenced and analyzed for binding properties either as pools of display-phage or as individual clones.

Two phases of selection were performed, each consisting of three rounds of binding and elution. Phase I selection used the phase I library (variegated residues 13, 16, 17, 18, and 19) which went through three rounds of binding and elution against a target protease giving rise to a subpopulation of clones. The R_(f) DNA derived from this selected subpopulation was used to generate the Phase II library (addition of variegated residues 31, 32, 34 and 39). The 1.8×10⁷ independent transformants were obtained for each of the phase II libraries. The phase II libraries underwent three further rounds of binding and elution with the same target protease giving rise to the final selectants.

Following two phases of selection against human plasma kallikrein-agarose beads a number (10) of the final selection display-phage were sequenced. The amino-acid sequences are shown in Table 2, entries KBPconl through KKII/3190 C.

Table 23 shows that KkII/3(D) is a highly specific inhibitor of human Kallikrein. Phage that display the LACI-K1 derivative KkII/3(D) bind to Kallikrein beads at least 50-times more than it binds to other protease targets.

Preliminary measurements indicate that KKII/3#6 is a potent inhibitor of pKA with K_(i) probably less than 500 pM.

Expression, Purification and Kinetic Analysis.

The three isolates KKII/3#6, KK2/#11, and KK2/#13 were recloned into a yeast expression vector. The yeast expression vector is derived from pMFalpha8 (KURJ82 and MIYA85). The LACI variant genes were fused to part of the matα 1 gene, generating a hybrid gene consisting of the matα 1 promoter-signal peptide and leader sequence-fused to the LACI variant. The cloning site is shown in Table 24. Note that the correctly processed LACI-K1 variant protein should be as detailed in Table 2 with the addition of residues glu-ala-ala-glu (SEQ ID NO: 70) to the N-terminal met (residue 1 in Table 2). Expression in S. cerevisiae gave acceptable yield typical of this system. Yeast-expressed LACI (kunitz domain 1), BPTI and LACI variants: KKII/3#6, KK2/#11, and KK2/#13 were purified by affinity chromatography using trypsin-agarose beads.

For larger-scale production, Pichia pastoris is a preferred host. The most preferred production system in P. pastoris is the alcohol oxidase system. Others have produced a number of proteins in the yeast Pichia pastoris. For example, Vedvick et al. (VEDV91) and Wagner et al. (WAGN92) produced aprotinin from the alcohol oxidase promoter with induction by methanol as a secreted protein in the culture medium at ≈1 mg/ml. Gregg et al. (GREG93) have reviewed production of a number of proteins in P. pastoris. Table 1 of GREG93 shows proteins that have been produced in P. pastoris and the yields.

All references, including those to U.S. and foreign patents or patent applications, and to nonpatent disclosures, are hereby incorporated by reference in their entirety.

                                      TABLE 1                                      __________________________________________________________________________     Sequence of whole LACI:                                                        1   5      5       5      5      5                                             __________________________________________________________________________      1 MIYTMKKVHA                                                                             LWASVCLLLN                                                                             LAPAPLNAds                                                                            eedeehtiit                                                                            dtelpplklM                                     51                                                                               HSFCAFKADD                                                                             GPCKAIMKRF                                                                             FFNIFTRQCE                                                                            EFIYGGCEGN                                                                            QNRFESLEEC                                    101                                                                               KKMCTRDnan                                                                             riikttlqqee                                                                            kpdfcfleed                                                                            pgicrgyitr                                                                            yfynnqtkqd                                    151                                                                               erfkyggclg                                                                             nmnnfetlee                                                                             cknicedgpn                                                                            gfqvdnygtq                                                                            lnavnnsltp                                    201                                                                               qstkvpslfe                                                                             fhgpswcltp                                                                             adrglcrane                                                                            nrfyynsvig                                                                            kcrpfkysgc                                    251                                                                               ggnennftsk                                                                             qeclrackkg                                                                             fiqriskggl                                                                            iktkrkrkkq                                                                            rvkiayeeif                                    301                                                                               vknm   (SEQ ID NO. 18)                                                      __________________________________________________________________________      The signal sequence (1-28) is uppercase and underscored                        LACIK1 is uppercase                                                            LACIK2 is underscored                                                          LACIK3 is bold                                                           

TABLE 2 is below.

                  TABLE 3                                                          ______________________________________                                         Summary of first selection of LACI-K1 domains for binding to pKA.                                    Library    Preferred                                     BPTI #   (Lac I)      Residues   Residues                                      ______________________________________                                         13       P            LHPR       HP                                            16       A            AG         AG                                            17       I            FYLHINA    NSA                                                                 SCPRTVD                                                                        G                                                        18       M            all        HL                                            19       K            LWQMKAG    QLP                                                                 SPRTVE                                                   31       E            EQ         E                                             32       E            EQ         EQ                                            34       I            all        STI                                           39       E            all        GEA                                           ______________________________________                                    

                                      TABLE 2                                      __________________________________________________________________________     Sequences of Kunitz domains, some of which inhibit human pKA.                           Amino-acid sequence                                                                 1111111111222222222233333333334444444444555555555                Ident    1234567890123456789012345678901234567890123456789012345678                                                              SEQ ID                       __________________________________________________________________________                                                       NO.                          BPTI     RPDECLEPPYTGPCKARIIRYEYNAKAGLCQTFVYGGCRAKRNNEKSAEDCMRTCGGA                                                              SEQ ID NO. 2                 LACI-K1  mhsfcafkaddgpckaimkrfffniftrqceefiyggcegnqnrfesleeckkmctrd                                                              SEQ ID NO. 3                 KBPcon1  mhsfcafkaddgHckaNHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 4                 KKII/3#1 mhsfcafkaddgHckASLPrfffniftrqcEEfIyggcEgnqnrfesleeckkmctrd                                                              SEQ ID NO. 5                 KKII/3#2 mhsfcafkaddgPckANHLrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 6                 KKII/3#3 mhsfcafkaddgHckANHQrfffniftrqcEEfTyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 7                 KKII/3#4 mhsfcafkaddgHckANHQrfffniftrqcEQfTyggcAgnqnrfesleeckkmctrd                                                              SEQ ID NO. 8                 KKII/3#5 mhsfcafkaddgHckASLPrfffniftrqcEEfIyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 9                 KKII/3#6 mhsfcafkaddgHckANHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 10                KKII/3#7 mhsfcafkaddgHckANHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 11                KKII/3#8 mhsfcafkaddgHckANHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 12                KKII/3#9 mhsfcafkaddgHckANHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 13                KKII/3#10                                                                               mhsfcafkaddgHckGAHLrfffniftrqcEEfIyggcEgnqnrfesleeckkmctrd                                                              SEQ ID NO. 14                KKI/3(a) mhsfcafkaddgRckGAHLrfffniftrqceefiyggcegnqnrfesleeckkmctrd                                                              SEQ ID NO. 15                KKI/3(b) mhsfcafkaddgPckAIHLrfffniftrqceefiyggcegnqnrfesleeckkmctrd                                                              SEQ ID NO. 16                KKI/3#C  mhsfcafkaddgHckANHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 17                KK2/#13  mhsfcafkaDGgRcRGAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 19                KK2/#14  mhsfcafkaDGgRcRGAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 20                KK2/#5   mhsfcafkaDDgPcRAAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 21                KK2/#11  mhsfcafkaDDgPcRAAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 22                KK2/#1   mhsfcafkaDVgRcRGAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 23                KK2/#4   mhsfcafkaDVgRcRGAQPrFffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 24                KK2/#6   mhsfcafkaDDgScRAAHLrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 25                KK2/#10  mhsfcafkaEGgScRAAHQrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 26                KK2/#8   mhsfcafkaDDgPcRGAHLrFffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 27                KK2/#3   mhsfcafkaDDgHcRGALPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 28                KK2/#9   mhsfcafkaDSgNcRGNLPrFffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 29                KK2/#7   mhsfcafkaDSgRcRGNHQrFffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 30                KK2/#12  mhsfcafkaDGgRcRAIQPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 31                KK2con1  mhsfcafkaDDgRcRGAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                                                              SEQ ID NO. 32                Human LACI-K2                                                                           KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG                                                              SEQ ID NO. 33                DKI-1.2.1                                                                               kpdfcfleedGgRcrgAHPrWfynnqtkqceEfSyggcGgnmnnfetleecknicedg                                                              SEQ ID NO. 34                Human LACI-K3                                                                           GPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG                                                              SEQ ID NO. 35                DKI-1.3.1                                                                               gpswcltpadDgPcraAHPrfyynsvigkcEpfSysgcggnennftskqeclrackkg                                                              SEQ ID NO. 36                Human collagen α3                                                                 ETDICKLPKDEGRCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV                                                              SEQ ID NO. 37                KuDom                                                                          DKI-2.1  etdicklpkdegtcrAAHlkwyydpntkscaEfSyggcggnenkfgsqkecekvcapv                                                              SEQ ID NO. 38                TFPI-2 DOMAIN 1                                                                         NAEICLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRI                                                              SEQ ID NO. 39                DKI-3.1.1                                                                               naeicllpldGgpcraAHlryyydrytqscEqfSyggcegnannfytweacddacwri                                                              SEQ ID NO. 40                tfpi-2 DOMAIN 2                                                                         VPKVCRLQVS-                              SEQ ID NO. 41                              VDDQCEGSTAKYFFNLSSMTCEKFFSGGCHRNR-                                                                   IENRFPDEATCMGFCAPK                         DKI-3.2.1                                                                               vpkvcrlqvs-                              SEQ ID NO. 42                              vddqcRAAHPkyffnlssmtceEffsggchrnr-                                                                   ienrfpdeatcmgfcapk                         TFPI-2 DOMAIN 3                                                                         IPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKA                                                              SEQ ID NO. 43                DKI-3.3.1                                                                               ipsfcyspkdegHcRaAHQryyfnpryrtcdaftytgcggndnnfvsredckracaka                                                              SEQ ID NO. 44                HUMAN ITI-K1                                                                            KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTV                                                              SEQ ID NO. 45                DKI-4.1.1                                                                               kedscqlgyDagpcRgAHPryfyngtsmacetfSyggcGgngnnfvtekeclqtcrtv                                                              SEQ ID NO. 46                Human ITI-K2                                                                            TVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP                                                              SEQ ID NO. 47                DKI-4.2.1                                                                               tvaacnlpiDDgpcraAHqlwafdavkgkcEEfSyggcEgngnkfysekecreycgvp                                                              SEQ ID NO. 48                DKI-4.2.2                                                                               tvaacnlpiDDgpcraAHqRwafdavkgkcEEfSyggcqgngnkfysekecreycgvp                                                              SEQ ID NO. 49                HUMAN    VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA                                                              SEQ ID NO. 50                PROTEASE                                                                       NEXIN-II                                                                       DKI-5.1  vrevcseqaetgpcraAHPrwyfdvtegkcEEfSyggcggnrnnfdteeycmavcgsa                                                              SEQ ID NO. 51                HKI B9 domain                                                                           LPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFLRKEKCEKFCKFT                                                              SEQ ID NO. 53                DKI-6.1  lpnvcafpmeDgpcRAAHPrwffnfetgeceEfayggcggnsnnflrkekcekfckft                                                              SEQ ID NO. 54                DKI-7.1  rpdfcleppEtgpcRaAHPryfynakaglcEEfvyggcGakrnnfksaedcmrtcgga                                                              SEQ ID NO.                   __________________________________________________________________________                                                       55                      

                  TABLE 8                                                          ______________________________________                                         Binding Data for Selected Kallikrein-binding Display-Phage.                    Display-Phage(a)                                                                            Fraction Bound(b)                                                                           Relative Binding(c)                                  ______________________________________                                         LACI         4.2 × 10.sup.-6                                                                       1.0                                                  BPTI         2.5 × 10.sup.-5                                                                       6.0                                                  KKI/3(a)     3.2 × 10.sup.-3                                                                       761                                                  KKI/3(b)     2.2 × 10.sup.-3                                                                       524                                                  KKII/3#5     3.9 × 10.sup.-3                                                                       928                                                  KKII/3#6     8.7 × 10.sup.-3                                                                       2071                                                 ______________________________________                                          (a) Clonal isolates of displayphage. LACIK1 is the parental molecule, BPT      (bovine pancreatic trypsin inhibitor) is a control and KKII/3 (5 and 6)        and KKI/3 (a and b) were selected by binding to the target protease,           kallikrein.                                                                    (b) The number of pfu's eluted after a binding experiment as a fraction o      the input number (10.sup.10 pfu's).                                            (c) Fraction bound relative to the parental displayphage, LACIK1.        

                  TABLE 9                                                          ______________________________________                                         Conservative and Semiconservative substitutions                                Initial           Conservative                                                                               Semi-conservative                                AA type                                                                               Category   substitution                                                                               substitution                                     ______________________________________                                         A      Small non- G, S, T     N, V, P, (C)                                            polar or                                                                       slightly                                                                       polar                                                                   C      free SH    A, M, L, V, I                                                                              F, G                                                    disulfide  nothing     nothing                                          D      acidic,    E, N, S, T, Q                                                                              K, R, H, A                                              hydrophilic                                                             E      acidic,    D, Q, S, T, N                                                                              K, R, H, A                                              hydrophilic                                                             F      aromatic   W, Y, H, L, M                                                                              I, V, (C)                                        G      Gly-only   nothing     nothing                                                 conformation                                                                   "normal"   A, S, N, T  D, E, H, I, K, L, M,                                    conformation           Q, R, V                                          H      amphoteric Y, F, K, R  L, M, A, (C)                                            aromatic                                                                I      aliphatic, V, L, M, A  F, Y, W, G (C)                                          branched β                                                                carbon                                                                  K      basic      R, H        Q, N, S, T, D, E, A                              L      aliphatic  M, I, V, A  F, Y, W, H, (C)                                  M      hydrophobic                                                                               L, I, V, A  Q, F, Y, W, (C), (R),                                                          (K), (E)                                         N      non-polar  S, T, (D), Q,                                                                              K, R                                                    hydrophilic                                                                               A, G, (E)                                                    P      inflexible V, I        A, (C), (D), (E), F,                                                           H, (K), L, M, N, Q,                                                            (R), S, T, W, Y                                  Q      aliphatic  N, E, A, S, T,                                                                             M, L, K, R                                              plus amide D                                                            R      basic      K, Q, H     S, T, E, D, A,                                   S      hydrophilic                                                                               A, T, G, N  D, E, R, K                                       T      hydrophilic                                                                               A, S, G, N, V                                                                              D, E, R, K, I                                    V      aliphatic, I, L, M, A, T                                                                              P, (C)                                                  branched β                                                                carbon                                                                  W      aromatic   F, Y, H     L, M, I, V, (C)                                  Y      aromatic   F, W, H     L, M, I, V, (C)                                  ______________________________________                                    

Changing from A, F, H, I, L, M, P, V, W, or Y to C is semiconservative if the new cysteine remains as a free thiol.

Changing from M to E, R, K is semiconservative if the ionic tip of the new side group can reach the protein surface while the methylene groups make hydrophobic contacts.

Changing from P to one of K, R, E, or D is semiconservative if the side group is on or near the surface of the protein.

                  TABLE 14                                                         ______________________________________                                         Definition of a Kunitz Domain (SEQ ID NO. 52)                                   ##STR1##                                                                      ______________________________________                                         Where:                                                                         X1, X2, X3, X4, X58, X57, and X56 may be absent,                               X21 = Phe, Tyr, Trp,                                                           X22 = Tyr or Phe,                                                              X23 = Tyr or Phe,                                                              X35 = Tyr or Trp,                                                              X36 = Gly or Ser,                                                              X40 = Gly or Ala,                                                              X43 = Asn or Gly, and                                                          X45 = Phe or Tyr                                                               ______________________________________                                    

                  TABLE 15                                                         ______________________________________                                         Substitution to confer high affinity for pKA on KuDoms                         Position                                                                             Preferred    Allowed      Unlikely to work                               ______________________________________                                         10    Asp, Glu     Ala, Gly, Ser, Thr                                                                          Lys, Asn (Arg, Cys,                                                            Phe, His, Ile, Leu,                                                            Met, Pro, Gln, Val,                                                            Trp, Tyr)                                      11    Asp, Gly, Ser, Val                                                                          Glu, Leu, Met,                                                                              (Cys, Phe, His, Lys,                                               Asn, Ile, Ala, Thr!                                                                        Pro, Gln, Arg, Trp,                                                            Tyr)                                           12    Gly          (Other amino acids                                                             ONLY if C.sub.14 --C.sub.38                                                    disulfide replaced                                                             by other amino                                                                 acids.)                                                     13    Arg, His, Pro, Asn,                                                                          Thr, Ala, Gly, Lys,                                                                        Phe, Tyr, Cys, Leu,                                  Ser          Gin!         Ile, Val, Asp (Glu,                                                            Met, Trp)                                      14    Cys          (Other amino acids                                                             ONLY if C.sub.38 also                                                          changed.)                                                   15    Arg, Lys      Ala, Ser, Gly, Met,                                                                        (Cys, Asp, Glu,                                                   Asn, Gln!    Phe, His, Ile, Leu,                                                            Pro, Thr, Val, Trp,                                                            Tyr)                                           16    Ala, Gly      Ser, Asp, Asn!                                                                             (Cys, Glu, Phe, His,                                                           Ile, Lys, Leu, Met,                                                            Pro, Gln, Arg, Thr,                                                            Val, Trp, Tyr)                                 17    Ala, Asn, Ser, Ile                                                                           Gly, Val, Gln, Thr                                                                         Cys, Asp, Phe, His,                                                            Pro, Arg, Tyr, (Glu,                                                           Lys, Met, Trp)                                 18    His, Leu, Gln                                                                                Ala,        Cys, Asp, Glu, Phe,                                                            Gly, Ile, Lys, Met,                                                            Asn, Pro, Arg, Ser,                                                            Thr, Val, Trp, Tyr                             19    Pro, Gly, Leu                                                                                Asn, Ile!   Ala, Glu, Gly, Met,                                                            Arg, Ser, Thr, Val,                                                            Trp, (Cys, Asp, Phe,                                                           His, Tyr)                                      20    Arg          Leu, Ala, Ser, Lys,                                                                         (Cys, Glu, Phe,                                                   Gln, Val     Gly, His, Ile, Met,                                                            Asn, Pro, Thr, Trp,                                                            Tyr)                                           21    Trp, Phe      Tyr, His, Ile!                                                                             Cys, Leu (Ala, Asp,                                                            Glu, Gly, Lys, Met,                                                            Asn, Pro, Gln, Arg,                                                            Ser, Thr, Val)                                 31    Glu           Asp, Gln, Asn, Ser,                                                                        (Arg, Lys, Cys, Phe,                                              Ala, Val, Leu, Ile,                                                                         Gly, His, Met, Pro,                                               Thr!         Trp, Tyr)                                      32    Glu, Gln      Asp, Asn, Pro, Thr,                                                                        (Cys, Phe, His, Ile,                                              Leu, Ser, Ala, Gly,                                                                         Lys, Met, Arg, Trp,                                               Val!         Tyr)                                           33    Phe           Tyr!        other 18 excluded.                             34    Ser, Thr, Ile                                                                                Val, Ala, Asn, Gly,                                                                        Cys, Asp, Glu, Phe,                                               Leu!         His, Lys, Met, Pro,                                                            Gln, Arg, Trp, Tyr                             35    Tyr           Trp, Phe!   (other 17)                                     36    Gly          Ser, Ala     (other 17)                                     37    Gly          (Other amino-acid                                                              types allowed only                                                             if C.sub.14 --C.sub.38 replaced                                                by other types.)                                            38    Cys          (Other amino acids                                                             ONLY if C.sub.14 also                                                          changed.)                                                   39    Gly, Glu, Ala                                                                                Ser, Asp!   Other 15.                                      ______________________________________                                    

Under "Preferred", most highly preferred type are bold

Under "Allowed" are types not actually tested, but judged to be acceptable. Types shown in square brackets were allowed and not selected, but are so similar to types that were selected that the type is unlikely to abolish pKA binding. Such types are not preferred, but pKA-binding proteins could have such types.

Under "Unlikely to work", types shown outside parentheses have been tried and no isolates had that type; types in parentheses have not been tested, but are judged to be unsuitable from consideration of the types actually excluded.

                  TABLE 21                                                         ______________________________________                                         First Variegation of LACI-K1                                                   ______________________________________                                          ##STR2##                                                                       ##STR3##                                                                       ##STR4##                                                                       ##STR5##                                                                       ##STR6##                                                                       ##STR7##                                                                      ______________________________________                                    

The segment from NsiI to MluI gives 65,536 DNA sequences and 31,200 protein sequences. Second group of variegation gives 21,840 and 32,768 variants. This variegation can go in on a fragment having MluI and one of AgeI, BstBI, or XbaI ends. Because of the closeness between codon 42 and the 3restriction site, one will make a self-priming oligonucleotide, fill in, and cut with MluI and, for example, BstBI. Total variants are 2.726×10⁹ and 8.59×10⁹.

The DNA sequence has SEQ ID NO. 56.

The amino acid sequence has SEQ ID NO. 57.

                  TABLE 23                                                         ______________________________________                                         Specificity Results                                                            Protein                                                                        displayed                                                                              Immobilized enzyme tested                                              on M13                                 Trypsin,                                gIIIp   Plasmin  Thrombin pKA   Trypsin                                                                               2 washes                                ______________________________________                                         LACI-K1 1        1        1     1      1                                       KkII/3(D)                                                                              3.4      1.5      196.  2.     1.4                                     BPTI    88.      1.1      1.7   0.3    .8                                      ______________________________________                                    

Numbers refer to relative binding of phage display clones compared to the parental phage display.

The KkII/3(D)(Kallikrein) clone retains the parental molecule's affinity for trypsin. KkII/3(D) was selected for binding to pKA.

                                      TABLE 24                                     __________________________________________________________________________     Mat α S. cerevisiae expression vectors:                                  __________________________________________________________________________     Matα1(Mfα8)                                                         ##STR8##                                                                      Matα2 (after introduction of a linker into StuI-cut DNA)                  ##STR9##                                                                      Matα-LACI-K1, amino acids: SEQ ID NO. 62, DNA: SEQ ID NO. 63              ##STR10##                                                                      ##STR11##                                                                      ##STR12##                                                                      ##STR13##                                                                      ##STR14##                                                                      ##STR15##                                                                     __________________________________________________________________________

We expect that Matα pre sequence is cleaved before GLU_(a) --ALA_(b)

                                      TABLE 27                                     __________________________________________________________________________     High specificity plasma Kallikrein inhibitors                                  __________________________________________________________________________     LACI-K1 (SEQ ID NO. 3)                                                         MHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD                     KKII/3#7 (SEQ ID NO. 11)                                                       mhsfcafkaddgHckANHQrfffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                     KKII/3#7-K15A (SEQ ID NO. 64)                                                  mhsfcafkaddghcAanhqrfffniftrqceefsyggcggnqnrfesleeckkmctrd                     KK2/#13-R15A (SEQ ID NO. 65)                                                   mhsfcafkaDGgRcAGAHPrWffniftrqcEEfSyggcGgnqnrfesleeckkmctrd                     KK2/#11-R15S (SEQ ID NO. 66)                                                   mhsfcafkaddgpcSaahprwffniftrqceefsyggcggnqnrfesleeckkmctrd                     __________________________________________________________________________

                                      TABLE 49                                     __________________________________________________________________________             Residue Number                                                                 10 11 12                                                                               13 14                                                                               15                                                                               16 17 18                                                                               19 20                                                                               21                                         __________________________________________________________________________     LACI-K1 D  D  G P  C K A  I  M K  R F                                          Consensus of                                                                           d  d  g H  c k A  N  H Q  r f                                          KKII/3                                                                         selectants                                                                     KK Library #2   FS                                                                             YC                                                                        NI   LP        NI                                                           NK AD   HR        AD QL                                                                               QL   FW                                                 DE ST g IT c KR                                                                               AG ST HP                                                                               HP r CL                                                    GV   NV        GV R R                                                               AD                                                                             G                                                              KK2/#13 -- G  --                                                                               -- --                                                                               --                                                                               -- -- --                                                                               -- --                                                                               --                                         KK2/#14 -- G  --                                                                               -- --                                                                               --                                                                               -- -- --                                                                               -- --                                                                               --                                         KK2/#5  -- -- --                                                                               P  --                                                                               --                                                                               A  -- --                                                                               -- --                                                                               --                                         KK2/#11 -- -- --                                                                               P  --                                                                               --                                                                               A  -- --                                                                               -- --                                                                               --                                         KK2/#1  -- V  --                                                                               -- --                                                                               --                                                                               -- -- --                                                                               -- --                                                                               --                                         KK2/#4  -- V  --                                                                               -- --                                                                               --                                                                               -- -- Q -- --                                                                               F                                          KK2/#6  -- -- --                                                                               S  --                                                                               --                                                                               A  -- --                                                                               L  --                                                                               --                                         KK2/#10 E  G  --                                                                               S  --                                                                               --                                                                               A  -- --                                                                               Q  --                                                                               --                                         KK2/#8  -- -- --                                                                               P  --                                                                               --                                                                               -- -- --                                                                               L  --                                                                               F                                          KK2/#3  -- -- --                                                                               H  --                                                                               --                                                                               -- -- L -- --                                                                               --                                         KK2/#9  -- S  --                                                                               N  --                                                                               --                                                                               -- N  L -- --                                                                               F                                          KK2/#7  -- S  --                                                                               -- --                                                                               --                                                                               -- N  --                                                                               Q  --                                                                               F                                          KK2/#12 -- G  --                                                                               -- --                                                                               --                                                                               A  I  Q -- --                                                                               --                                         Consensus #2                                                                           D  D  g R  c R G  A  H P  r W                                          __________________________________________________________________________

                  TABLE 750                                                        ______________________________________                                         DNA that embodies Library KKF.                                                  ##STR16##                                                                      ##STR17##                                                                      ##STR18##                                                                     The RsrII and BspHI sites found in the parental LACI-K1                        display gene (Table 6) are not present in Library KKF.                         There are 1,536,000 amino-acid sequences and 4,194,304 DNA                     sequences. Met.sub.18 and Lys.sub.19 are not allowed in Library                ______________________________________                                         KKF.                                                                      

Citations:

ADEL86: Adelman et al., Blood (1986) 68(6)1280-1284.

ALBR83a: Albrecht et al, Hoppe-Seyler's Z Physiol Chem (1983), 364:1697-1702.

ALBR83b: Albrecht et al., Hoppe-Seyler's ZPhysiol Chem (1983), 364:1703-1708.

ANBA88: Anba et al, Biochimie (1988) 70(6)727 -733.

ANGL87: Angliker et al., Biochem J (1987) 241(3)871-5.

AUER88: Auerswald et al., Bio Chem Hoppe-Seyler (1988), 369(Supplement):27-35.

BALD85: Balduyck et al., Biol Chem Hoppe-Seyler (1985) 366:9-14.

BANE90: Baneyx & Georgiou, J Bacteriol (1990) 172(1)491-494.

BANE91: Baneyx & Georgiou, J Bacteriol (1991) 173(8)2696-2703.

BERN93: Berndt et al., Biochemistry (1993) 32:4564-70.

BHOO92: Bhoola et al., Pharmacological Reviews (1992) 44(1)1-80.

BROW91: Browne et al., GeneBank entry M74220.

BROZ90: Broze et al., Biochemistry (1990) 29:7539-7546.

COLM87: Colman et al., Editors, Hemostasis and Thrombosis, Second Edition, 1987, J. B. Lippincott Company, Philadelphia, Pa.

COLM87a: Colman et al., Chapter 1 of COLM87.

DAVI79: Davis et al, U.S. Pat. No. 4,179,337 (1979).

DENN94a: Dennis & Lazarus, J Biological Chem (1994) 269:22129-22136.

DENN94b: Dennis & Lazarus, J Biological Chem (1994) 269:22137-22144.

EIGE90: Eigenbrot et al., Protein Engineering (1990), 3(7)591-598.

ELLI92: Ellis et al., Ann N Y Acad Sci (1992) 667:13-3 1.

FIDL94: Fidler & Ellis, Cell (1994) 79:185-188.

FRAE89: Fraedrich et al., Thorac Cardiovasc Surg (1989) 37(2)89-91.

GARD93: Gardell, Toxicol Pathol (1993) 21(2)190-8.

GIRA89: Girard et al., Nature (1 989), 338:518-20.

GlRA91: Girard et al., J BIOL. CHEM. (1991) 266:5036-5041.

HOOV93: Hoover et al., Biochemistry (1993)32:1093643.

HORT91: Hortin & Trimpe, J Biol Chem (1991) 266(11)6866-71.

HYNE90: Hynes et al., Biochemistry (1990), 29:10018-10022.

KEMP88b: Kemp & Bowen, Tetrahedron Letts (1988) 29:5077-5080.

KIDO88: Kido et al, J Biol Chem (1988), 263:18104-7.

KIDO90: Kido et al., Biochem & Biophys Res Comm (1990), 167(2)716-21.

KLIN91: Kline et al., Biochem Biophys Res Commun (1991) 177(3)1049-55.

LASK80: Laskowski & Kato, Ann Rev Biochem (1980), 49:593-626.

LEAT91: Leatherbarrow & Salacinski, Biochemistry (1991) 30(44)10717-21.

LOHM93: Lohrnann & J Marshall, Refract Corneal Surg (1993) 9(4)300-2.

LUCA83: Lucas et al., J Biological Chem (1983) 258(7)4249-56.

MANN87: Mann & Foster, Chapter 10 of COLM87.

MARC85: Advanced Organic Chemistry, Third Edition March, J, John Wiley and Sons, New York, 1985; ISBN 0-471-88841-9.

MIYA85: Miyajima et al., Gene (1985) 37:155-161.

NEUH89: Neuhaus et al., Lancet (1989) 2(8668)924-5.

NOVO89: Novotny et al., J. BIOL. CHEM. (1989) 264:18832-18837.

PARK86: Park & Tulinsky, Biochemistry (1986) 25(14)3977-3982.

PUTT89: Putternan, Acta Chir Scand (1989) 155(6-7)367.

ROBB87: Robbins, Chapter 21 of COLM87

SCHE67: Schechter & Berger. Biochem Biophys Res Commun (1967) 27:157-162.

SCHE68: Schechter & Berger. Biochem Biophys Res Commun (1968) 32:898-902.

SCHM87: Schmaier et al., Chapter 2 in COLM87.

SCHN86: Schnabel et al., Biol Chem Hoppe-Seyler (1986), 367:1167-76.

SHER89: Sheridan et al., Dis Colon Rectum (1989) 32(6)505-8.

TIAN92: Tian et al., Int J Pept Protein Res (1992)40(2)119-26.

VAND91: van der Logt et al, BIOCHEMISTRY (1991) 30:1571-1577.

VAND92: van Diji et al., EMBO J (1992) 11(8)2819-2828.

VARA83: Varadi & Patthy, Biochemistry (1983) 22:2440-2446.

VARA84: Varadi & Patthy, Biochemistry (1984) 23:2108-2112.

WILS93: Wilson et al., Tetrahedron (1993)49(17)3655-63.

WUNT88: Wun et al., J. BIOL. CHEM. (1988) 263:6001-6004.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 70                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:8 amino acids                                                       (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        IleValGlyGlyThrAsnSerSer                                                       15                                                                             (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLysAla                               151015                                                                         IleMetLysArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         SerLeuProArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLysAla                               151015                                                                         AsnHisLeuArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheThrTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGln                               202530                                                                         PheThrTyrGlyGlyCysAlaGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         SerLeuProArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheIleTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysGly                               151015                                                                         AlaHisLeuArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyArgCysLysGly                               151015                                                                         AlaHisLeuArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLysAla                               151015                                                                         IleHisLeuArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysLysAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:304 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       MetIleTyrThrMetLysLysValHisAlaLeuTrpAlaSerValCys                               151015                                                                         LeuLeuLeuAsnLeuAlaProAlaProLeuAsnAlaAspSerGluGlu                               202530                                                                         AspGluGluHisThrIleIleThrAspThrGluLeuProProLeuLys                               354045                                                                         LeuMetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLys                               505560                                                                         AlaIleMetLysArgPhePhePheAsnIlePheThrArgGlnCysGlu                               65707580                                                                       GluPheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPheGluSer                               859095                                                                         LeuGluGluCysLysLysMetCysThrArgAspAsnAlaAsnArgIle                               100105110                                                                      IleLysThrThrLeuGlnGlnGluLysProAspPheCysPheLeuGlu                               115120125                                                                      GluAspProGlyIleCysArgGlyTyrIleThrArgTyrPheTyrAsn                               130135140                                                                      AsnGlnThrLysGlnCysGluArgPheLysTyrGlyGlyCysLeuGly                               145150155160                                                                   AsnMetAsnAsnPheGluThrLeuGluGluCysLysAsnIleCysGlu                               165170175                                                                      AspGlyProAsnGlyPheGlnValAspAsnTyrGlyThrGlnLeuAsn                               180185190                                                                      AlaValAsnAsnSerLeuThrProGlnSerThrLysValProSerLeu                               195200205                                                                      PheGluPheHisGlyProSerTrpCysLeuThrProAlaAspArgGly                               210215220                                                                      LeuCysArgAlaAsnGluAsnArgPheTyrTyrAsnSerValIleGly                               225230235240                                                                   LysCysArgProPheLysTyrSerGlyCysGlyGlyAsnGluAsnAsn                               245250255                                                                      PheThrSerLysGlnGluCysLeuArgAlaCysLysLysGlyPheIle                               260265270                                                                      GlnArgIleSerLysGlyGlyLeuIleLysThrLysArgLysArgLys                               275280285                                                                      LysGlnArgValLysIleAlaTyrGluGluIlePheValLysAsnMet                               290295300                                                                      (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       MetHisSerPheCysAlaPheLysAlaAspGlyGlyArgCysArgGly                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       MetHisSerPheCysAlaPheLysAlaAspGlyGlyArgCysArgGly                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysArgAla                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysArgAla                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       MetHisSerPheCysAlaPheLysAlaAspValGlyArgCysArgGly                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       MetHisSerPheCysAlaPheLysAlaAspValGlyArgCysArgGly                               151015                                                                         AlaGlnProArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlySerCysArgAla                               151015                                                                         AlaHisLeuArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       MetHisSerPheCysAlaPheLysAlaGluGlyGlySerCysArgAla                               151015                                                                         AlaHisGlnArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysArgGly                               151015                                                                         AlaHisLeuArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysArgGly                               151015                                                                         AlaLeuProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       MetHisSerPheCysAlaPheLysAlaAspSerGlyAsnCysArgGly                               151015                                                                         AsnLeuProArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       MetHisSerPheCysAlaPheLysAlaAspSerGlyArgCysArgGly                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       MetHisSerPheCysAlaPheLysAlaAspGlyGlyArgCysArgAla                               151015                                                                         IleGlnProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyArgCysArgGly                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       LysProAspPheCysPheLeuGluGluAspProGlyIleCysArgGly                               151015                                                                         TyrIleThrArgTyrPheTyrAsnAsnGlnThrLysGlnCysGluArg                               202530                                                                         PheLysTyrGlyGlyCysLeuGlyAsnMetAsnAsnPheGluThrLeu                               354045                                                                         GluGluCysLysAsnIleCysGluAspGly                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       LysProAspPheCysPheLeuGluGluAspGlyGlyArgCysArgGly                               151015                                                                         AlaHisProArgTrpPheTyrAsnAsnGlnThrLysGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnMetAsnAsnPheGluThrLeu                               354045                                                                         GluGluCysLysAsnIleCysGluAspGly                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       GlyProSerTrpCysLeuThrProAlaAspArgGlyLeuCysArgAla                               151015                                                                         AsnGluAsnArgPheTyrTyrAsnSerValIleGlyLysCysArgPro                               202530                                                                         PheLysTyrSerGlyCysGlyGlyAsnGluAsnAsnPheThrSerLys                               354045                                                                         GlnGluCysLeuArgAlaCysLysLysGly                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:36:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       GlyProSerTrpCysLeuThrProAlaAspAspGlyProCysArgAla                               151015                                                                         AlaHisProArgPheTyrTyrAsnSerValIleGlyLysCysGluPro                               202530                                                                         PheSerTyrSerGlyCysGlyGlyAsnGluAsnAsnPheThrSerLys                               354045                                                                         GlnGluCysLeuArgAlaCysLysLysGly                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:37:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                       GluThrAspIleCysLysLeuProLysAspGluGlyThrCysArgAsp                               151015                                                                         PheIleLeuLysTrpTyrTyrAspProAsnThrLysSerCysAlaArg                               202530                                                                         PheTrpTyrGlyGlyCysGlyGlyAsnGluAsnLysPheGlySerGln                               354045                                                                         LysGluCysGluLysValCysAlaProVal                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                       GluThrAspIleCysLysLeuProLysAspGluGlyThrCysArgAla                               151015                                                                         AlaHisLeuLysTrpTyrTyrAspProAsnThrLysSerCysAlaGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGluAsnLysPheGlySerGln                               354045                                                                         LysGluCysGluLysValCysAlaProVal                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       AsnAlaGluIleCysLeuLeuProLeuAspTyrGlyProCysArgAla                               151015                                                                         LeuLeuLeuArgTyrTyrTyrAspArgTyrThrGlnSerCysArgGln                               202530                                                                         PheLeuTyrGlyGlyCysGluGlyAsnAlaAsnAsnPheTyrThrTrp                               354045                                                                         GluAlaCysAspAspAlaCysTrpArgIle                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:40:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                       AsnAlaGluIleCysLeuLeuProLeuAspGlyGlyProCysArgAla                               151015                                                                         AlaHisLeuArgTyrTyrTyrAspArgTyrThrGlnSerCysGluGln                               202530                                                                         PheSerTyrGlyGlyCysGluGlyAsnAlaAsnAsnPheTyrThrTrp                               354045                                                                         GluAlaCysAspAspAlaCysTrpArgIle                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:61 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                       ValProLysValCysArgLeuGlnValSerValAspAspGlnCysGlu                               151015                                                                         GlySerThrGluLysTyrPhePheAsnLeuSerSerMetThrCysGlu                               202530                                                                         LysPhePheSerGlyGlyCysHisArgAsnArgIleGluAsnArgPhe                               354045                                                                         ProAspGluAlaThrCysMetGlyPheCysAlaProLys                                        505560                                                                         (2) INFORMATION FOR SEQ ID NO:42:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:61 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                       ValProLysValCysArgLeuGlnValSerValAspAspGlnCysArg                               151015                                                                         AlaAlaHisProLysTyrPhePheAsnLeuSerSerMetThrCysGlu                               202530                                                                         GluPhePheSerGlyGlyCysHisArgAsnArgIleGluAsnArgPhe                               354045                                                                         ProAspGluAlaThrCysMetGlyPheCysAlaProLys                                        505560                                                                         (2) INFORMATION FOR SEQ ID NO:43:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                       IleProSerPheCysTyrSerProLysAspGluGlyLeuCysSerAla                               151015                                                                         AsnValThrArgTyrTyrPheAsnProArgTyrArgThrCysAspAla                               202530                                                                         PheThrTyrThrGlyCysGlyGlyAsnAspAsnAsnPheValSerArg                               354045                                                                         GluAspCysLysArgAlaCysAlaLysAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:44:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                       IleProSerPheCysTyrSerProLysAspGluGlyHisCysArgAla                               151015                                                                         AlaHisGlnArgTyrTyrPheAsnProArgTyrArgThrCysAspAla                               202530                                                                         PheThrTyrThrGlyCysGlyGlyAsnAspAsnAsnPheValSerArg                               354045                                                                         GluAspCysLysArgAlaCysAlaLysAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:45:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                       LysGluAspSerCysGlnLeuGlyTyrSerAlaGlyProCysMetGly                               151015                                                                         MetThrSerArgTyrPheTyrAsnGlyThrSerMetAlaCysGluThr                               202530                                                                         PheGlnTyrGlyGlyCysMetGlyAsnGlyAsnAsnPheValThrGlu                               354045                                                                         LysGluCysLeuGlnThrCysArgThrVal                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:46:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                       LysGluAspSerCysGlnLeuGlyTyrAspAlaGlyProCysArgGly                               151015                                                                         AlaHisProArgTyrPheTyrAsnGlyThrSerMetAlaCysGluThr                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlyAsnAsnPheValThrGlu                               354045                                                                         LysGluCysLeuGlnThrCysArgThrVal                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:47:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                       ThrValAlaAlaCysAsnLeuProIleValArgGlyProCysArgAla                               151015                                                                         PheIleGlnLeuTrpAlaPheAspAlaValLysGlyLysCysValLeu                               202530                                                                         PheProTyrGlyGlyCysGlnGlyAsnGlyAsnLysPheTyrSerGlu                               354045                                                                         LysGluCysArgGluTyrCysGlyValPro                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:48:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                       ThrValAlaAlaCysAsnLeuProIleAspAspGlyProCysArgAla                               151015                                                                         AlaHisGlnLeuTrpAlaPheAspAlaValLysGlyLysCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGluGlyAsnGlyAsnLysPheTyrSerGlu                               354045                                                                         LysGluCysArgGluTyrCysGlyValPro                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:49:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                       ThrValAlaAlaCysAsnLeuProIleAspAspGlyProCysArgAla                               151015                                                                         AlaHisGlnArgTrpAlaPheAspAlaValLysGlyLysCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlnGlyAsnGlyAsnLysPheTyrSerGlu                               354045                                                                         LysGluCysArgGluTyrCysGlyValPro                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:50:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                       ValArgGluValCysSerGluGlnAlaGluThrGlyProCysArgAla                               151015                                                                         MetIleSerArgTrpTyrPheAspValThrGluGlyLysCysAlaPro                               202530                                                                         PhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPheAspThrGlu                               354045                                                                         GluTyrCysMetAlaValCysGlySerAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:51:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                       ValArgGluValCysSerGluGlnAlaGluThrGlyProCysArgAla                               151015                                                                         AlaHisProArgTrpTyrPheAspValThrGluGlyLysCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnArgAsnAsnPheAspThrGlu                               354045                                                                         GluTyrCysMetAlaValCysGlySerAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:52:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                       XaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaGlyXaaCysXaaXaa                               151015                                                                         XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaCysXaaXaa                               202530                                                                         PheXaaXaaXaaGlyCysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa                               354045                                                                         XaaXaaCysXaaXaaXaaCysXaaXaaXaa                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:53:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                       LeuProAsnValCysAlaPheProMetGluLysGlyProCysGlnThr                               151015                                                                         TyrMetThrArgTrpPhePheAsnPheGluThrGlyGluCysGluLeu                               202530                                                                         PheAlaTyrGlyGlyCysGlyGlyAsnSerAsnAsnPheLeuArgLys                               354045                                                                         GluLysCysGluLysPheCysLysPheThr                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:54:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                       LeuProAsnValCysAlaPheProMetGluAspGlyProCysArgAla                               151015                                                                         AlaHisProArgTrpPhePheAsnPheGluThrGlyGluCysGluGlu                               202530                                                                         PheAlaTyrGlyGlyCysGlyGlyAsnSerAsnAsnPheLeuArgLys                               354045                                                                         GluLysCysGluLysPheCysLysPheThr                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:55:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                       ArgProAspPheCysLeuGluProProGluThrGlyProCysArgAla                               151015                                                                         AlaHisProArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGluGlu                               202530                                                                         PheValTyrGlyGlyCysGlyAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:56:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:186 bases                                                           (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:other nucleic acid                                          (A) DESCRIPTION:synthetic DNA fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                       GCCGAGATGCATTCCTTCTGCGCCTTCAAGGCTGATGATGGTCNTTGTAA50                           AGSTNNTNNSNNGCGTTTCTTCTTCAACATCTTCACGCGTCAGTGCSAGS100                          AATTCNNSTACGGTGGTTGTNNSGGTAACCAGAACCGGTTCGAATCTCTA150                          GAGGAATGTAAGAAGATGTGCACTCGTGACGGCGCC186                                        (2) INFORMATION FOR SEQ ID NO:57:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:62 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                       AlaGluMetHisSerPheCysAlaPheLysAlaAspAspGlyXaaCys                               151015                                                                         LysXaaXaaXaaXaaArgPhePhePheAsnIlePheThrArgGlnCys                               202530                                                                         XaaXaaPheXaaTyrGlyGlyCysXaaGlyAsnGlnAsnArgPheGlu                               354045                                                                         SerLeuGluGluCysLysLysMetCysThrArgAspGlyAla                                     505560                                                                         (2) INFORMATION FOR SEQ ID NO:58:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:4 amino acids                                                       (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                       LysArgProArg                                                                   (2) INFORMATION FOR SEQ ID NO:59:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:13 bases                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:other nucleic acid                                          (A) DESCRIPTION:synthetic DNA fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                       AAAAGGCCTCGAG13                                                                (2) INFORMATION FOR SEQ ID NO:60:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:14 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                       LysArgGluAlaAlaGluProTrpGlyAlaXaaXaaLeuGlu                                     1510                                                                           (2) INFORMATION FOR SEQ ID NO:61:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:42 bases                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:other nucleic acid                                          (A) DESCRIPTION:synthetic DNA fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                       AAAAGGGAAGCGGCCGAGCCATGGGGCGCCTAATAGCTCGAG42                                   (2) INFORMATION FOR SEQ ID NO:62:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:66 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                       LysArgGluAlaAlaGluMetHisSerPheCysAlaPheLysAlaAsp                               151015                                                                         AspGlyProCysLysAlaIleMetLysArgPhePhePheAsnIlePhe                               202530                                                                         ThrArgGlnCysGluGluPheIleTyrGlyGlyCysGluGlyAsnGln                               354045                                                                         AsnArgPheGluSerLeuGluGluCysLysLysMetCysThrArgAsp                               505560                                                                         GlyAla                                                                         65                                                                             (2) INFORMATION FOR SEQ ID NO:63:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:210 bases                                                           (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:other nucleic acid                                          (A) DESCRIPTION:synthetic DNA fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                       AAAAGGGAAGCGGCCGAGATGCATTCCTTCTGCGCTTTCAAAGCTGATGA50                           CGGTCCGTGTAAAGCTATCATGAAACGTTTCTTCTTCAACATTTTCACGC100                          GTCAGTGCGAGGAATTCATTTACGGTGGTTGTGAAGGTAACCAGAACCGG150                          TTCGAATCTCTAGAGGAATGTAAGAAGATGTGCACTCGTGACGGCGCCTA200                          ATAGCTCGAG210                                                                  (2) INFORMATION FOR SEQ ID NO:64:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyHisCysAlaAla                               151015                                                                         AsnHisGlnArgPhePhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:65:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                       MetHisSerPheCysAlaPheLysAlaAspGlyGlyArgCysAlaGly                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:66:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                       MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysSerAla                               151015                                                                         AlaHisProArgTrpPhePheAsnIlePheThrArgGlnCysGluGlu                               202530                                                                         PheSerTyrGlyGlyCysGlyGlyAsnGlnAsnArgPheGluSerLeu                               354045                                                                         GluGluCysLysLysMetCysThrArgAsp                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:67:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:97 bases                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:other nucleic acid                                          (A) DESCRIPTION:synthetic DNA fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                       CCTCCTATGCATTCCTTCTGCGCCTTCAAGGCTRASRNTGGTNNTTGTAR50                           AGSTRNTCNSCNSCGTTKSTTCTTCAACATCTTCACGCGTTCCCTCC97                              (2) INFORMATION FOR SEQ ID NO:68:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:23 bases                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:other nucleic acid                                          (A) DESCRIPTION:synthetic DNA fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                       GGAGGGAACGCGTGAAGATGTTG23                                                      (2) INFORMATION FOR SEQ ID NO:69:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:28 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                       MetHisSerPheCysAlaPheLysAlaXaaXaaGlyXaaCysXaaXaa                               151015                                                                         XaaXaaXaaArgXaaPhePheAsnIlePheThrArg                                           2025                                                                           (2) INFORMATION FOR SEQ ID NO:70:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:4 amino acids                                                       (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                       GluAlaAlaGlu                                                                   14                                                                             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We claim:
 1. A kallikrein inhibiting protein which comprises a non-naturally naturally occurring Kunitz domain, wherein, at each of the residues of said domain corresponding to the below identified residues of BPTI, one of the following allowed amino acids is found:

    ______________________________________                                         BPTI                                                                           residue #      Allowed Amino Acid                                              ______________________________________                                         10             Asp, Glu, Ala, Gly, Ser, Thr                                    11             Asp, Gly, Ser, Val, Glu, Leu,                                                  Met, Asn, Ile, Ala, Thr                                         12             Gly, and, if residue 14 or 38                                                  is not Cys, any conservative or                                                semi-conservative substitution                                                 for a "normal" conformation Gly                                                as defined in Table 9                                           13             Arg, His, Pro, Asn, Ser, Thr,                                                  Ala, Gly, Lys, Gln                                              14             Cys, and, if residue 38 is not                                                 Cys, any conservative or semi-                                                 conservative substitution for                                                  Cys                                                             15             Arg, Lys, Ala, Ser, Gly, Met,                                                  Asn, Gln                                                        16             Ala, Gly, Ser, Asp, Asn                                         17             Ala, Asn, Ser, Ile, Gly, Val,                                                  Gln, Thr                                                        18             His, Leu, Gln, Ala                                              19             Pro, Gln, Leu, Asn, Ile                                         20             Arg, Leu, Ala, Ser, Lys, Gln,                                                  Val                                                             21             Trp, Phe, Tyr, His, Ile                                         31             Glu, Asp, Gln, Asn, Ser, Ala,                                                  Val, Leu, Ile, Thr                                              32             Glu, Gln, Asp, Asn, Pro, Thr,                                                  Leu, Ser, Ala, Gly, Val                                         33             Phe, Tyr                                                        34             Ser, Thr, Ile, Val, Ala, Asn,                                                  Gly, Leu                                                        35             Tyr, Trp, Phe                                                   36             Gly, Ser, Ala                                                   37             Gly, and, if residue 14 or 38                                                  is not Cys, any conservative or                                                semi-conservative substitution                                                 for a "normal" conformation Gly                                                as defined in Table 9                                           38             Cys, and, if residue 14 is not                                                 Cys, any conservative or semi-                                                 conservative substitution for                                                  Cys                                                             39             Gly, Glu, Ala, Ser, Asp.                                        ______________________________________                                    


2. A method of treating a disorder attributable to excessive kallikrein activity which comprises administering, to a human or animal subject who would benefit therefrom, a kallikrein-inhibitory amount of the protein of claim
 1. 3. A method of assaying for the presence of kallikrein in a sample which method comprises: providing the protein of claim 1 in labeled or insolubilized form, contacting said protein with said sample, and determining whether a complex of said protein and kallikrein in the sample is formed.
 4. A method of purifying kallikrein from a mixture which comprises: providing the protein of claim 1 in insolubilized form, and contacting the mixture with said insolubilized protein so that kallikrein in the mixture is bound.
 5. A kallikrein inhibiting protein which comprises a non-naturally occurring Kunitz domain, wherein, at each of the residues corresponding to the below identified residues, one of the following allowed amino acids is found:

    ______________________________________                                         BPTI                                                                           residue #      Allowed Amino Acid                                              ______________________________________                                         10             Asp, Glu, Ala, Gly, Ser, Thr                                    11             Asp, Gly, Ser, Val, Glu, Leu,                                                  Met                                                             12             Gly, and, if residue 14 or 38                                                  is not Cys, or any conservative                                                semi-conservative substitution                                                 for a "normal" conformation Gly                                                as defined in Table 9                                           13             Arg, His, Pro, Asn, Ser                                         14             Cys, and, if residue 38 is not                                                 Cys, any conservative or semi-                                                 conservative substitution for                                                  Cys                                                             15             Arg, Lys                                                        16             Ala, Gly                                                        17             Ala, Asn, Ser, Ile                                              18             His, Leu, Gln                                                   19             Pro, Gln, Leu                                                   20             Arg, Leu, Ala, Ser, Lys, Gln,                                                  Val                                                             21             Trp, Phe                                                        31             Glu                                                             32             Glu, Gln                                                        33             Phe                                                             34             Ser, Thr, Ile                                                   35             Tyr                                                             36             Gly, Ser, Ala                                                   37             Gly, and, if residue 14 or 38                                                  not Cys, any conservative or                                                   semi-conservative substitution                                                 for a "normal" conformation Gly                                                as defined in Table 9                                           38             Cys, and, if residue                                                           corresponding to position 14 is                                                not Cys, any conservative or                                                   semi-conservative substitution                                  39             Gly, Glu, Ala.                                                  ______________________________________                                    


6. The protein of claim 2 wherein, the Kunitz domain is further characterized as follows:

    ______________________________________                                         BPTI                                                                           Residue No.        Allowed Residue                                             ______________________________________                                         10                 Asp, Glu                                                    11                 Asp, Gly, Ser, Val                                          12                 Gly                                                         14                 Cys                                                         20                 Arg                                                         36                 Gly                                                         37                 Gly                                                         38                 Cys.                                                        ______________________________________                                    


7. A method of preventing or treating a disorder attributable to excessive kallikrein activity which comprises administering, to a human or animal subject who would benefit therefrom, a kallikrein-inhibitory amount of the protein of claim
 5. 8. A method of assaying for the presence of kallikrein in a sample which method comprises: providing the protein of claim 5 in labeled or insolubilized form, contacting said protein with said sample and determining whether a complex of said protein and kallikrein in the sample is formed.
 9. A method of purifying kallikrein from a mixture which comprises: providing the protein of claim 5 in insolubilized form, and contacting the mixture with said insolubilized protein so that kallikrein in the mixture is bound.
 10. A plasma kallikrein inhibiting protein which comprises a sequence that is homologous to a reference sequence being selected from the group consisting of: KKII/3#1 (SEQ ID NO. 5), KKII/3#2 (SEQ ID NO. 6), KKII/3#3 (SEQ ID NO. 7), KKII/3#4 (SEQ ID NO. 8), KKII/3#5 (SEQ ID NO. 9), KKII/3#6 (SEQ ID NO. 10), KKII/3#7 (SEQ ID NO. 11), KKII/3#8 (SEQ ID NO. 12), KKII/3#9 (SEQ ID NO. 13), KKII/3#10 (SEQ ID NO. 15), KK2/#11 (SEQ ID NO. 22), KK2/#13 (SEQ ID NO. 19), KK2/#1 (SEQ ID NO. 23), KK2/#2, ! KK2/#3 (SEQ ID NO. 28), KK2/#4 (SEQ ID NO. 24), KK2/#6 (SEQ ID NO. 25), KK2/#7 (SEQ ID NO. 30), KK2/#8 (SEQ ID NO. 27), KK2/#9 (SEQ ID NO. 29) KK2/#10 (SEQ ID NO. 27), KK2/#12 (SEQ ID NO. 31), and KK2con1 (SEQ ID NO.32) as defined in Table
 2. 11. A method of treating a disorder attributable to excessive kallikrein activity which comprises administering, to a human or animal subject who would benefit therefrom, a kallikrein-inhibitory amount of the protein of claim
 10. 12. A method of assaying for the presence of kallikrein in a sample which method comprises: providing the protein of claim 10 in labeled or insolubilized form, contacting said protein with said sample, and determining whether a complex of said protein and kallikrein in the sample is formed.
 13. A method of purifying kallikrein from a mixture which comprises: providing the protein of claim 10 in insolubilized form, and contacting the mixture with said insolubilized protein so that kallikrein in the mixture is bound. 